CN106290603A - A kind of utilize Vavle switching method to detect the inorganic anion in plant, organic acid and the method for three kinds of phytochemicalss and application simultaneously - Google Patents

A kind of utilize Vavle switching method to detect the inorganic anion in plant, organic acid and the method for three kinds of phytochemicalss and application simultaneously Download PDF

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CN106290603A
CN106290603A CN201610575040.8A CN201610575040A CN106290603A CN 106290603 A CN106290603 A CN 106290603A CN 201610575040 A CN201610575040 A CN 201610575040A CN 106290603 A CN106290603 A CN 106290603A
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CN106290603B (en
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陈梅兰
吴琪琪
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Zhejiang Shuren University
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/08—Preparation using an enricher
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • G01N2030/201—Injection using a sampling valve multiport valves, i.e. having more than two ports

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Abstract

The invention discloses and a kind of utilize Vavle switching method to detect inorganic anion in plant, organic acid and the method for three kinds of phytochemicalss and application simultaneously.Sample is first leached liquid and brings in the C18 post being connected to high performance liquid chromatograph, owing to organic acid and anion do not retain in C18 post, by arranging the suitable Vavle switching time, organic acid and anion is made all to be switched in ion chromatography post, it is retained in C18 post three kinds of phytochemicalss to continue to be rushed out chromatographic column along with the drip washing of leacheate, detects by UV-detector.The anion and the organic acid that enter ion chromatographic column are rushed out chromatographic column along with alkalescence leacheate drip washing, detect with electric conductivity detector.So connect high performance liquid chromatograph by Vavle switching and can detect inorganic anion, organic acid and three kinds of phytochemicalss with ion chromatograph simultaneously.The stability in the large of this method, repeatability is preferable, and accurately and reliably, can be widely applied to food production, biological medicine etc. needs to detect the industry of active ingredient to determination data.

Description

A kind of utilize Vavle switching method to detect the inorganic anion in plant, organic acid simultaneously Method and application with three kinds of phytochemicalss
Technical field
The invention belongs to analytical chemistry field, be specifically related to a kind of utilize that Vavle switching method detects in plant simultaneously inorganic Anion, organic acid and the method for three kinds of phytochemicalss and application.
Background technology
At present, the inorganic anion in detection plant, organic acid generally use high performance anion exchange chromatography method (ion color Spectrometry), and the phytochemicalss such as scopoletin, rutin, Quercetin use the mode such as reversed phase high-performance liquid chromatography, spectrophotography to carry out Individually measure.But in analyzing test, it is often necessary to inorganic anion, organic acid and different phytochemicalss are surveyed simultaneously Fixed.If still using separately independent method for measuring, not only resulting in the waste of manpower financial capacity, and cannot be met some to inspection Survey ageing stronger application.Still do not have a kind of method the most reliably can measure above-mentioned substance content, therefore at present simultaneously Need a kind of inorganic anion, organic acid and method of various plants element that can simultaneously detect in plant of exploitation badly.
Summary of the invention
It is an object of the invention to solve problems of the prior art, and provide one to utilize Vavle switching method simultaneously Organic acid, inorganic anion and the method for three kinds of phytochemicalss in detection plant and application.Concrete technical scheme is as follows:
Utilize Vavle switching method to detect the organic acid in plant, inorganic anion and the method for three kinds of phytochemicalss, profit simultaneously It is used in conjunction high performance liquid chromatograph uv detection method with high performance anion exchange chromatography conductance detection to plant sample by Vavle switching method In product, organic acid, inorganic anion and scopoletin, rutin, three kinds of phytochemicalss of Quercetin carry out separation detection；After pre-treatment Plant sample is first leached liquid and brings in the C18 post being connected to high performance liquid chromatograph, organic acid and anion in C18 post not Retaining, during by arranging suitable Vavle switching, the acid of chien shih machine and anion are all switched in ion chromatography post, retain Three kinds of phytochemicalss in C18 post continue to be rushed out chromatographic column along with the drip washing of leacheate, detect by UV-detector；Enter from The sample of sub-chromatographic column is rushed out chromatographic column along with alkali liquor leacheate drip washing, detects with electric conductivity detector.
As preferably, plant sample pre-treating method is as follows: sample is placed at a temperature of 80 DEG C, grinds after drying 2 hours Mill, crosses the sieve of 0.5mm, accurately weighs the sample 0.0400g less than 0.5mm, be placed in 10mL volumetric flask, and accurately add 2.00mL ultra-pure water and 8.00mL methanol fully shake up；Arranging frequency is that 50KHz carries out supersound extraction, and Extracting temperature is 20 DEG C, The supersound extraction time is 1h, moves in 10mL polypropylene centrifuge tube, and under 2500r/min rotating speed, centrifugal 20min, takes supernatant mistake Sample introduction analysis after 0.45 μm microporous filter membrane.
Further, realize, by Vavle switching, the method that inorganic anion, organic acid and three kinds of phytochemicalss separate as follows: enter Before sample, six-way valve 1 is maintained at Load state, in the 20 μ L quantitative loop that sample manually sample introduction is loaded in six-way valve 1, is noted The redundant sample entered enters waste liquid；Six-way valve 2 is maintained at Inject state；After sample introduction completes, start changeover program, switch clematis stem Valve 1, starts sample introduction, and six-way valve 1 is in Inject state, and six-way valve 2 remains at Inject state, and leacheate is by quantitative loop Sample inject in C18 post, in C18 post without reserve, and three kinds of phytochemicalss are withed a hook at the end above for inorganic anion, organic acid, Separated；After starting changeover program 1.1min, switching six-way valve 2, make six-way valve 2 be in Load state, enriching column is rich Collection organic acid, anion, enrichment time is 2.5min；After being enriched with, switching six-way valve 2, makes six-way valve 2 come back to again Inject state, enriching column is connected with analytical column IonPac AS11-HC, the organic acid of separation and concentration, anion, simultaneously C18 post Separating plant element.
Further, in chromatography of ions: analytical column uses IonPac AS11-HC (4mm × 250mm), guard column uses IonPac AG11-HC (4mm × 50mm), enriching column uses IonPac TAC-ULP1 (5mm × 23mm), and chromatographic condition is as follows: Flow velocity 1.0mL/min, column temperature 30 DEG C；Gradient elution program: 0-18.6min, concentration is 0.8mmol/L KOH solution；18.6- 33.6min, concentration is 0.8-15mmol/L KOH solution；33.6-53.6min, concentration is 15mmol/L KOH solution；53.6- 68.6min, concentration is 15-50mmol/L KOH solution；68.6-68.7min, concentration is 50-0.8mmol/L KOH solution； 68.7-69.6min, concentration is 0.8mmol/L KOH solution.
In high performance liquid chromatography: C18 post uses Yi Lite Hypersil ODS analytical column (4.6mm × 250mm, particle diameter 5 μ M), flowing phase: methanol (C), water (D), flow velocity 1.0mL/min, column temperature 25 DEG C, wavelength 365nm；Sample size 20 μ L；Gradient elution Program: 0-12min, 20%-40%C, 80%-60%D；12-14min, 40%-70%C, 60%-30%D；14-22min, 70%-90%C, 30%-10%D；22-25min, 90%-20%C, 10%-80%D.
Said method can be applicable to detect in food production and biological medicine industry active ingredient.
The method of the present invention utilizes Vavle switching method that with high-efficiency anion, high performance liquid chromatography uv detection method is exchanged color Spectrum conductance detection is combined, can during one-time detection simultaneously to inorganic anion in plant sample, organic acid and Scopoletin, rutin, three kinds of phytochemicalss of Quercetin carry out separation detection.The present invention also has favorable reproducibility, detection limits low, precision Good advantage.
Accompanying drawing explanation
Fig. 1 is that Vavle switching device sample loads block diagram；In figure, between six-way valve difference hole, solid line represents connection, dotted line generation Table does not connects, lower same；
Fig. 2 is that Vavle switching device Yi Lite Hypersil ODS analytical column (C18 post) separates sample block diagram；
Fig. 3 is Vavle switching device enriching column enrichment anion and organic acid block diagram；
Fig. 4 is Vavle switching device anion, organic acid and phytochemicals analytical procedure figure simultaneously；
Fig. 5 is 50ppm organic acid standard mixed solution chromatography of ions figure；
Fig. 6 is 10ppm organic acid standard mixed solution liquid chromatogram；
Fig. 7 is second switching time and anion, organic acid peak area graph of a relation；
Fig. 8 is first switching time and anion, organic acid peak area graph of a relation；
Fig. 9 is 10ppm standard mixed solution chromatography of ions figure；Wherein number and be divided into representative: 1-lactic acid；2-acetic acid；3-third Acid；4-formic acid；5-Cl-；6-malic acid；7-SO4 2-；8-oxalic acid；9-PO4 3-；10-citric acid.
Figure 10 is 1ppm standard mixed solution liquid chromatogram；Wherein number and represent respectively: 1-scopoletin；2-rutin；3- Quercetin.
Figure 11 is Radix Glycyrrhizae sample ions chromatogram；Wherein number and represent respectively: 1-lactic acid；2-acetic acid；3-formic acid；4-Cl-； 5-malic acid；6-SO4 2-；7-oxalic acid；8-PO4 3-；9-citric acid.
Figure 12 is Radix Glycyrrhizae sample liquid chromatogram；Wherein number and represent respectively: 1-scopoletin；2-rutin；3-Quercetin.
Detailed description of the invention
Below in conjunction with accompanying drawing and case study on implementation, the present invention is described further.
The chromatography of inorganic anion, organic acid and phytochemicals, specifically comprises the following steps that
1, instrument and reagent
The inventive method based on Vavle switching apparatus structure as it is shown in figure 1, include pump, six-way valve 1, Yi Lite Hypersil ODS analytical column (C18 post), six-way valve 2, enriching column, guard column, analytical column, suppressor, electric conductivity detector, UV detector.Clematis stem Valve 1 carries out sample introduction by quantitative loop, and is connected with six-way valve 2 by Yi Lite Hypersil ODS analytical column, and enriching column is placed in In six-way valve 2, six-way valve 2 one tunnel is connected with UV detector, another road successively with guard column, analytical column, suppressor, Conductivity detection Device is connected.The waste liquid outlet of each detecting element is all connected with waste collecting device.
INSTRUMENT MODEL and parameter that part is used are specific as follows: Thermo ICS 5000 ion chromatograph (Conductivity detection Device, six-way valve, (external aqueous mode uses and wears peace gradient pump GP50, wears regulating the spleen and stomach state ASRS 300 (4mm) self-circulating electrical suppressor Company limited), KOH leacheate automatic generator EG40, Chromeleon 6.8 chromatographic work station, it is Chinese that Sai Mo flies your science and technology of generation Company limited)；(gradient mixes Agilent 1200series high performance liquid chromatograph for high pressure six-way injection valve, online degassing instrument Instrument and UV-Vis detector, Co., Ltd in Agilent Technologies)；KQ-300GVD type three frequency constant-temperaturenumerical-control numerical-control ultrasonic cleaning device (Kunshan Ultrasonic Instruments Co., Ltd.)；BS-224S type electronic analytical balance (Sai Duolisi scientific instrument (Beijing) limited public affairs Department)；Column oven (sky, Shenzhen is mechanical & electronic equipment corporation, Ltd).
Reagent is as follows: methanol (chromatographically pure, world TEDIA company limited of the U.S.), lactic acid, acetic acid, propanoic acid, formic acid, Cl-、 Malic acid, SO4 2-, oxalic acid, PO4 3-, citric acid (analytical pure, purchased from Huadong Medicine Co., Ltd.), scopoletin (Aladdin try Agent), rutin (>=98%), Quercetin (biochemical reagents) (purchased from Huadong Medicine Co., Ltd.), experimental water is ultra-pure water (Millipore, Molsheim, France, resistivity 18.2M Ω cm).
Laboratory sample: Radix Glycyrrhizae, Fructus Amomi Rotundus and Semen Euryales sample, purchased from local supermarket.
2, chromatographic condition
The separation chromatography condition of organic acid and anion and detection: Sai Mo flies ICS5000 ion chromatograph band Conductivity detection Instrument analytical column IonPac AS11-HC (4mm × 250mm) (representing pillar model parameter in bracket, lower same)+guard column IonPac AG11-HC (4mm × 50mm) and enriching column IonPac TAC-ULP1 (5mm × 23mm), flow velocity 1.0mL/min, column temperature 30 DEG C. Gradient elution program: 0-18.6min concentration is 0.8mmol/LKOH solution, 18.6-33.6min concentration is 0.8-15mmol/ LKOH solution, 33.6-53.6min concentration is 15mmol/LKOH solution, and 53.6-68.6min concentration is that 15-50mmol/LKOH is molten Liquid, 68.6-68.7min concentration is 50-0.8mmol/LKOH solution, and 68.7-69.6min concentration is 0.8mmol/LKOH solution. Eluent gradient table is shown in Table 1.Typical chromatogram is shown in Fig. 9.
Table 1 chromatography of ions eluent gradient table
The separation chromatography condition of phytochemicals and detection: Agilent 1200 high liquid chromatograph of liquid band UV-detector, according to profit Special Hypersil ODS analytical column (4.6mm × 250mm, particle diameter 5 μm), flow phase: methanol (being designated as C), water (being designated as D), flow velocity 1.0mL/min, column temperature 25 DEG C, wavelength 365nm；Sample size 20 μ L.Gradient elution program: 0-12min, 20%-40%C, 80%- 60%D；12-14min, 40%-70%C, 60%-30%D；14-22min, 70%-90%C, 30%-10%D；22-25min, 90%-20%C, 10%-80%D.Eluent gradient table is shown in Table 2.Typical chromatogram is shown in Figure 10.
The efficient liquid phase eluent gradient table of table 2
3, the preparation of standard solution
Standard Stock solutions: Cl-、SO4 2-、PO4 3-, lactic acid, acetic acid, propanoic acid, formic acid, malic acid, oxalic acid, citric acid is by excellent Level pure reagent is configured to the stock solution that concentration is 1000mg/L, and scopoletin, rutin, Quercetin are configured to concentration by Aladdin reagent For the stock solution of 1000mg/L, it is stored in 4 DEG C of refrigerators standby.
Standard mixed solution: pipette a certain amount of above-mentioned Standard Stock solutions respectively in the volumetric flask of 10mL, with ultrapure Water constant volume, join each solute concentration be 0.08mg/L, 0.40mg/L, 1.00mg/L, 2.00mg/L, 10.00mg/L, The mixed standard solution series of 50.00mg/L is standby.Pipette a certain amount of Cl the most respectively-、SO4 2-、PO4 3-, lactic acid, acetic acid, Propanoic acid, formic acid, malic acid, oxalic acid, citric acid Standard Stock solutions to 10mL volumetric flask in, use ultra-pure water constant volume, join respectively Solute concentration is the organic acid standard mixed solution of 10.00mg/L.
4, sample pre-treatments
Sample is placed at a temperature of 80 DEG C, grinds after drying 2 hours, cross the sieve of 0.5mm, accurately weigh and be less than The sample 0.0400g of 0.5mm, is placed in 10mL volumetric flask, and accurately addition 2.00mL ultra-pure water and 8.00mL methanol fully shake Even；Arranging frequency is that 50KHz carries out supersound extraction, and Extracting temperature is 20 DEG C, after supersound extraction 1h, moves into 10mL polypropylene centrifuge Guan Zhong, under 2500r/min rotating speed, centrifugal 20min, takes supernatant and crosses sample introduction analysis after 0.45 μm microporous filter membrane.
5, experimental procedure
Before sample introduction, six-way valve 1 is maintained at Load state, and sample manually sample introduction is loaded into the quantitative loop in six-way valve 1 In (20 μ L), the redundant sample injected enters waste liquid；Six-way valve 2 is maintained at Inject state, so can avoid in system The foreign ion that may be present gathering on enriching column, such as Fig. 1.After sample introduction completes, start changeover program (0min), switch six Logical valve 1, starts sample introduction, and six-way valve 1 is in Inject state, and six-way valve 2 remains at Inject state, and leacheate is by quantitative loop In sample inject in Yi Lite Hypersil ODS analytical column, organic acid and anion are analyzed at Yi Lite Hypersil ODS , and three kinds of phytochemicalss are withed a hook at the end above in post without reserve, are separated, such as Fig. 2.After 1.1min, switch six-way valve 2, Make six-way valve 2 be in Load state, enriching column enrichment organic acid and anion, be enriched with 2.5min, such as Fig. 3.After being enriched with, then Secondary switching six-way valve 2, makes six-way valve 2 come back to Inject state, and enriching column is connected with analytical column IonPac AS11-HC, point From organic acid and the anion of enrichment, Yi Lite Hypersil ODS analytical column separating plant element simultaneously, such as Fig. 4.
6, the selection of switching time
Directly connect electric conductivity detector (connection of unrestraint device) below at Yi Lite Hypersil ODS analytical column (C18 post) With UV-Vis detector, observe 10ppm organic acid standard mixed solution goes out peak situation, as Fig. 5,6.
In order to obtain switching time accurately, prevent that enrichment time is too short or long causes enrichment not exclusively, 50ppm is marked Quasi-mixed solution carries out a series of test.First, under conditions of being 1.4min first switching time, test respectively When 3.2min, 3.3min, 3.4min, 3.5min, 3.6min, 3.7min standard mixed solution go out peak situation, obtaining each has Machine acid, time and the peak area relation curve of anion, such as Fig. 7.From this curve chart, during 3.6min, concentration effect is best, Therefore selecting 3.6min is second switching time.Then, under conditions of being 3.6min second switching time, test respectively Standard mixed solution when 0.8min, 0.9min, 1.0min, 1.1min, 1.2min, 1.3min, 1.4min, 1.5min, 1.6min Go out peak situation, obtain time and the peak area relation curve of each organic acid, anion, such as Fig. 8.From this curve chart, During 1.1min, concentration effect is best, and therefore selecting 1.1min is first switching time.
7, the range of linearity, repeatability and detection limit
Under chromatographic condition above, by 0.08mg/L, 0.40mg/L, 2.00mg/L, 10.00mg/L, 50.00mg/L system The sample introduction analysis successively of row mixed standard solution, each concentration balance measures 3 times, and data are by, after test of difference, taking 3 gained The meansigma methods of peak area, with peak area (Y) as vertical coordinate, material concentration (X) is the standard curve that abscissa sets up 13 kinds of materials. Calculating minimum detectability with 3 times of baseline noises (S/N=3), when investigating repeatability, the hybrid standard with concentration as 2.00mg/L is molten Liquid is continuous sample introduction 8 times (n=8), computational methods precision under similarity condition.Its linear equation, correlation coefficient, the range of linearity, Detection limit and precision are shown in Table 3.
Table 3 range of linearity, repeatability and detection limit
8, actual sample detection and the response rate of method
Actual Radix Glycyrrhizae, Fructus Amomi Rotundus, Semen Euryales sample are detected according to after the pre-treatment described in step 4, and adds the denseest The standard mixed solution of degree, carries out recovery test, the results are shown in Table shown in 4～6, and its response rate all meets the requirements.Actual sample Chromatography of ions figure is shown in that Figure 11 (organic acid and anion chromatographic figure) high-efficient liquid phase chromatogram is shown in Figure 12 (phytochemicals sample chromatogram figure).
Table 4 Radix Glycyrrhizae sample recovery of standard addition
Note: "-" shows not measure.
Table 5 Fructus Amomi Rotundus sample recovery of standard addition
Note: "-" shows not measure.
Table 6 Semen Euryales sample recovery of standard addition
Note: "-" shows not measure.

Claims (5)

1. utilize Vavle switching method to detect the inorganic anion in plant, organic acid and a method for three kinds of phytochemicalss simultaneously, It is characterized in that, utilize Vavle switching method combination high performance liquid chromatograph uv detection method with ion chromatograph conductance detection to planting In thing sample, inorganic anion, organic acid and scopoletin, rutin, three kinds of phytochemicalss of Quercetin carry out separation detection, detection method For: the plant sample after pre-treatment is first leached liquid and brings in the C18 post being connected to efficient liquid phase instrument, organic acid and anion Not retaining in C18 post, during by arranging suitable Vavle switching, chien shih organic acid and anion are all switched to chromatography of ions In analytical column, it is retained in C18 post three kinds of phytochemicalss and continues to be rushed out chromatographic column along with the drip washing of leacheate, use ultraviolet detection Device detects；The anion and the organic acid that enter ion chromatographic column are rushed out chromatographic column along with alkalescence leacheate drip washing, examine with conductance Survey device detects.
2. the method for claim 1, it is characterised in that plant sample pre-treating method is as follows: sample is placed in 80 DEG C At a temperature of, grind after drying 2 hours, cross the sieve of 0.5mm, accurately weigh the sample 0.0400g less than 0.5mm, be placed in In 10mL volumetric flask, and accurately addition 2.00mL ultra-pure water and 8.00mL methanol fully shake up；Arranging frequency is that 50KHz surpasses Sound extracts, and Extracting temperature is 20 DEG C, after supersound extraction 1h, moves in 10mL polypropylene centrifuge tube, under 2500r/min rotating speed from Heart 20min, takes supernatant and crosses sample introduction analysis after 0.45 μm microporous filter membrane.
3. method as claimed in claim 2, it is characterised in that realize inorganic anion, organic acid and three kinds by Vavle switching The method that phytochemicals separates is as follows: before sample introduction, six-way valve 1 is maintained at Load state, and sample manually sample introduction is loaded into clematis stem In 20 μ L quantitative loop in valve 1, the redundant sample injected enters waste liquid；Six-way valve 2 is maintained at Inject state；Sample introduction completes After, starting changeover program, switch six-way valve 1, start sample introduction, six-way valve 1 is in Inject state, and six-way valve 2 remains at Inject state, the sample in quantitative loop is injected in C18 post by leacheate, organic acid and anion in C18 post without reserve, and Three kinds of phytochemicalss are withed a hook at the end above, are separated；After starting changeover program 1.1min, switch six-way valve 2, make six-way valve 2 are in Load state, and enriching column enrichment organic acid and anion, enrichment time is 2.5min；After being enriched with, again switch six Logical valve 2, makes six-way valve 2 come back to Inject state, and enriching column is connected with analytical column IonPac AS11-HC, separation and concentration Organic acid and anion, simultaneously C18 post separating plant element.
4. method as claimed in claim 3, it is characterised in that in chromatography of ions: analytical column uses IonPac AS11-HC (4mm × 250mm), guard column uses IonPac AG11-HC (4mm × 50mm), and enriching column uses IonPac TAC-ULP1 (5mm × 23mm), chromatographic condition is as follows: flow velocity 1.0mL/min, column temperature 30 DEG C；Gradient elution program: 0-18.6min, concentration is 0.8mmol/L KOH solution；18.6-33.6min, concentration is 0.8-15mmol/L KOH solution；33.6-53.6min, concentration is 15mmol/L KOH solution；53.6-68.6min, concentration is 15-50mmol/L KOH solution；68.6-68.7min, concentration is 50-0.8mmol/L KOH solution；68.7-69.6min, concentration is 0.8mmol/L KOH solution；
In high performance liquid chromatography: C18 post uses Yi Lite Hypersil ODS analytical column (4.6mm × 250mm, particle diameter 5 μm), stream Dynamic phase: methanol (C), water (D), flow velocity 1.0mL/min, column temperature 25 DEG C, wavelength 365nm；Sample size 20 μ L；Gradient elution program: 0-12min, 20%-40%C, 80%-60%D；12-14min, 40%-70%C, 60%-30%D；14-22min, 70%- 90%C, 30%-10%D；22-25min, 90%-20%C, 10%-80%D.
5. the application of a method as claimed in claim 1, it is characterised in that be applied in food production and biological medicine industry Detection active ingredient.
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CN107589188A (en) * 2017-09-04 2018-01-16 浙江树人学院 The separation of Vavle switching technology coupled HPLC while the method for detecting the carbohydrate in beverage, sweetener and preservative
CN108760941A (en) * 2018-08-06 2018-11-06 通标标准技术服务(上海)有限公司 A kind of pump of bivalve three highly effective liquid phase chromatographic system
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