CN102590190A - Method for measuring chlorogenic acid by using Fe<2+>-H2O<2-> methylene blue chemical luminescence system - Google Patents
Method for measuring chlorogenic acid by using Fe<2+>-H2O<2-> methylene blue chemical luminescence system Download PDFInfo
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- CN102590190A CN102590190A CN2012100227529A CN201210022752A CN102590190A CN 102590190 A CN102590190 A CN 102590190A CN 2012100227529 A CN2012100227529 A CN 2012100227529A CN 201210022752 A CN201210022752 A CN 201210022752A CN 102590190 A CN102590190 A CN 102590190A
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Abstract
The invention discloses a method for measuring chlorogenic acid by using a Fe<2+>-H2O<2-> methylene blue chemical luminescence system. H2SO4 is used as the solvent to prepare an ammonium ferrous sulfate solution. Redistilled water is used as the solvent to prepare a methylene blue solution, a hydrogen dioxide solution, a chlorogenic acid standard solution and a sample chlorogenic acid solution. The prepared ammonium ferrous sulfate solution and the chlorogenic acid standard solution are subjected to three-way mixing respectively by a main driving pump and the mixed solutions are input into a flow injection chemiluminescence analyzer. The prepared methylene blue solution and the hydrogen dioxide solution are injected into a flow-through cell by an auxiliary driving pump and then the chemical luminescence intensity I is measured and the data are recorded; and a standard curve equation is as follows: I=-4.0125x+2047, wherein x is counted with mu g/ml, the concentration is in the range of 1-500mu g/ml, and R2=0.9944. According the above operation process, the chemical luminescence intensity of the chlorogenic acid in the sample is measured and then substituted into the standard curve equation, and the content of the chlorogenic acid in the sample is counted by the standard curve equation that: x=(2047-I)/4.0125, wherein x is counted with mu g/ml. The detection method is simple, the cost is low and the conventional detection can be performed online.
Description
Technical field
The present invention relates to the assay method of chlorogenic acid, specifically is to use Fe
2+-H
2O
2-methylene blue chemical luminous system is measured the method for chlorogenic acid.The invention belongs to the research field such as physiology, plant biological active substance, food, health products, medicine of phytochemistry, natural product chemistry, Food Chemistry, analytical chemistry, organic chemistry, biological chemistry, Secondary Metabolism of Plant material.
Background technology
Chlorogenic acid (Chlorogenic acid) be caffeotannic acid again, chemistry 3-caffeoyl guinic acid by name.Chlorogenic acid is a kind of polyphenol compound; By a part caffeic acid (Caffeic acid) and a part chinic acid (Quinic acid; 1-hydroxyl six hydrogen gallic acids) depside that forms of condensation dehydration; Molecular structure is seen Figure of description, and it is a kind of phenylpropyl alcohol chlorins compound that plant produces through shikimic acid pathway in the aerobic respiration process.
Chlorogenic acid has stronger pharmacologically active, has broad-spectrum antiseptic, antiviral and antimycotic effect; Be mainly used in clearing heat and detoxicating, wind-heat dissipating, swollen sore, the larynx numbness erysipelas treated of pain; The warm blood fulminant dysentery, treatment of diseases such as anemopyretic cold can reduce cholesterol level in the blood; Strengthen the body defensive enginery, have higher medical value.
Along with the increase of microbiotic abuse and bad reaction, the cry of developing the Chinese medicine with good antibacterial effect is increasingly high.Chlorogenic acid more and more receives the world of medicine and pays close attention to as the significant Chinese medicine of antibacterial action.Therefore, the detection to chlorogenic acid content also becomes essential important component.
The method of qualitative and quantitative analysis chlorogenic acid mainly contains AAS (Sun Yan refined 2007 for Yuan Hua, Deng Liang), high performance liquid chromatography (plum forests 2007 both at home and abroad at present; Zhu Qingfeng, Chen Meirong etc. 2011), thin-layered chromatography (Nie Songliu, the Meng, lintel 2010), (Yang Xin etc. 1999 for capillary electrophoresis; Lv Yuanqi, Wu Chunhua, Yuan notable refined 2004) and chemoluminescence method (He Caixia, Lu Ming firm 1997; Zhang Hongying, Xu Xiangdong, Shi Hongmei etc. 2011).
There is certain defective in existing detection method: AAS is more consuming time, and poor reproducibility; The high performance liquid chromatography instrument is somewhat expensive, needs the professional and technical personnel simultaneously, and cultivating outstanding detection teacher needs long time, and special-purpose in addition chromatographically pure moving phase and chromatographic column are somewhat expensive; The used chemical reagent of thin-layered chromatography and capillary electrophoresis is many, and complex steps is suitable for the purifying of small sample; The chemoluminescence method that Zhang Hongying, Xu Xiangdong etc. do need be used more valuable Ag (III); The more inconvenient widespread use of the chemoluminescence method interfering material that He Caixia, Lu Minggang etc. do.Chemiluminometry is because it is highly sensitive, the range of linearity is wide, instrument and equipment is simple, more and more receives people's favor.Adopt luminol-potassium ferricyanide luminescence system and luminol-KIO
4The method document that system is measured chlorogenic acid has report, but Fe
2+-H
2O
2The mensuration that-methylene blue chemical luminous system is used for chlorogenic acid is not reported.
Summary of the invention
The objective of the invention is the defective to existing detection method, provide a kind of operation simpler, it is low to detect cost, the Fe of the good linearity of typical curve
2+-H
2O
2-methylene blue chemical luminous system is measured the method for chlorogenic acid.
Concrete steps are:
(1) preparation reagent: with 1～20 * 10
-3Mol/L H
2SO
4As solvent, preparation 1～20 * 10
-3L ferrous ammonium sulfate solution (the Fe of mol/L
2+); As solvent, prepare 1～20 * 10 with redistilled water respectively
-5The methylene blue solution of mol/L, concentration of volume percent are 0.1～5% oxydol, chlorogenic acid standard solution and the sample solution of chlorogenic acid that concentration is 0.001～10mg/mL.
(2) setting of portable injection chemiluminescence detector: the l ferrous ammonium sulfate solution (Fe that active efflux prepares step (1) respectively
2+) and the chlorogenic acid standard solution carry out threeway with the flow velocity of 30～60r/min through corresponding pipeline and mix, the input injected chemical luminescence analyzer that flows; The methylene blue solution that secondary moving pump prepares step (1) and oxydol inject flow cells with the flow velocity of 50～80r/min through 16 logical introduction valves, annotate 10～20 a seconds time of appearance, and each liquid mixing detects its chemiluminescence intensity I, record data.Above-mentioned test process carries out at 35 ℃ ± 1 ℃ constant temperature;
(3) make typical curve equation: I=-4.0125x+2047, wherein x is in μ g/ml, and concentration is at 1～500 μ g/ml scope, R
2=0.9944;
(4) sample determination of chlorogenic acid: according to step (2) operating process, the chemiluminescence intensity of specimen chlorogenic acid, substitution typical curve equation, calculation sample chlorogenic acid contents: x=(2047-I)/4.0125, wherein x is in μ g/ml.
Under optimized conditions, the concentration of chlorogenic acid standard solution is at 1～500 μ g/m
LWith the linear I=-4.0125x+2047 of luminous intensity I, wherein x is in μ g/ml, R in the scope
2=0.9944, this shows and can utilize this method that chlorogenic acid is carried out quantitative test.
The present invention compares with existing method, and the instrument and equipment of this assay method is simple, and is easy to operate, analyzes fast, and it is low, highly sensitive to detect cost, good reproducibility, and the range of linearity is wide, can onlinely carry out routine property mensuration, is easy to be applied in industry or the agricultural production.The present invention is a kind of method of online detection chlorogenic acid.Generally detected chlorogenic acid sample needs 20～30 minutes in the past, and the time required for the present invention is only within 5 minutes, and is rapider than existing other detection method, therefore can be applied to the suitability for industrialized production of extraction and the purifying and the crystallization of chlorogenic acid; Be rich in chlorogenic acid cultivation of plants and breeding; The content detection of chlorogenic acid in food, health products, medicine.
Description of drawings
Accompanying drawing is the molecular structure of chlorogenic acid.
Embodiment
Embodiment:
1. instrument and equipment
IFFM-E type portable injection chemiluminescence analyser (Xi'an Rui Mai Electronics Co., Ltd.).Sampling in the The whole analytical process, notes appearance, experimental data collection and processing are accomplished by IFFM-E type portable injection chemiluminescence analyser under the Windows XP system.
2. material and reagent
2.1 material:
Chlorogenic acid standard items: Nat'l Pharmaceutical & Biological Products Control Institute;
Chlorogenic acid sample: Xi'an damp bio tech ltd difficult to understand.
2.2 preparation reagent: use 0.01mol/L H
2SO
4As solvent, preparation 2.2 * 10
-3L ferrous ammonium sulfate solution (the Fe of mol/L
2+); As solvent, prepare 6.4 * 10 with redistilled water
-5The methylene blue solution of mol/L, concentration of volume percent 0.5% oxydol, concentration are 10mg/mL chlorogenic acid standard solution and sample solution of chlorogenic acid; Use concentration to go out the serial solution of a plurality of variable concentrations of interval 1～1000 μ g/mL of concentration again, use when manufacturing typical curve as 10mg/mL chlorogenic acid standard solution preparation.
3. the setting of portable injection chemiluminescence detector: active efflux is respectively with the l ferrous ammonium sulfate solution (Fe for preparing
2+) and the chlorogenic acid standard solution carry out threeway with the flow velocity of 45r/min through corresponding pipeline and mix, the input injected chemical luminescence analyzer that flows; Secondary moving pump with the methylene blue solution for preparing and oxydol with the flow velocity of 55r/min through 16 logical introduction valves injection flow cells; Annotate the 15 seconds time of appearance, each liquid mixes, and detects its chemiluminescence intensity; Record data are made typical curve equation (I=-4.0125x+2047); Repeat the aforesaid operations process then, the chemiluminescence intensity of specimen chlorogenic acid (I=1653.0), substitution typical curve equation, calculation sample chlorogenic acid contents (x=98.2 μ g/ml).Above-mentioned test process carries out at 35 ℃ ± 1 ℃ constant temperature.
4. test and interpretation of result
4.1 the optimization of test condition
4.1.1 stream parameters of choice
The influence of flow velocity: in Flow Injection Analysis, flow velocity is the key factor that sensitivity is measured in influence.Flow velocity can cause maximum luminous before flow cell too slowly; Flow velocity can cause maximum luminous after flow cell too soon; Under the prerequisite of fixing the shortest valve pond distance; Revolution to the major-minor peristaltic pump is investigated in 10～90r/min scope, and when the revolution of confirming best major-minor pump was respectively 45r/min (2.25ml/min) and 55r/min (2.75ml/min), I was maximum.
Other parameter: runner pipe (polyfluortetraethylene pipe) internal diameter is 0.8mm, and the valve pond is apart from being 12cm, and the photomultiplier negative high voltage is 800V.
4.1.2 single factor experiment is confirmed the concentration range of reaction medium
Fe
2+Concentration: Fe has been investigated in test
2+Concentration is 1.0 * 10
-4～5.0 * 10
-3To the influence of chemiluminescence intensity, the result shows: chemiluminescence intensity is with Fe in the mol/L scope
2+Concentration increases and increases, and works as Fe
2+Concentration surpasses 2.2 * 10
-3Luminous intensity tends towards stability during mol/L.Filter out Fe according to investigating the result
2+Concentration is 2.2 * 10
-3Mol/L.
Methylene blue concentration: test has been investigated methylene blue concentration 1.0 * 10
-5～1.0 * 10
-4The interior influence of mol/L scope to chemiluminescence intensity.The result shows: when low concentration, luminous intensity a little less than; Along with methylene blue concentration increases, luminous intensity also increases; When methylene blue concentration is 6.4 * 10
-5Luminous intensity reaches maximum during mol/L.Filtering out methylene blue concentration according to the investigation result is 6.4 * 10
-5Mol/L.
Hydrogen peroxide concentration: oxydol influence to chemiluminescence intensity in concentration of volume percent 0.1%～5% scope has been investigated in test; The result shows: along with the increase of hydrogen peroxide concentration; Luminous intensity increases rapidly, and luminous intensity reaches maximum when hydrogen peroxide concentration increases to 0.5%.The test and Selection hydrogen peroxide concentration is 0.5%.
Sulfuric acid concentration: tested sulfuric acid concentration 1.0 * 10
-4～1.0 * 10
-1To the influence of chemiluminescence intensity, the result shows: when sulfuric acid concentration is 1.0 * 10 in the mol/L scope
-2Luminous intensity reaches maximum during mol/L, and the test and Selection sulfuric acid concentration is 1.0 * 10
-2Mol/L.
4.2 typical curve, precision, repeatability, detectability and stability
4.2.1 the acquisition of typical curve equation
Under optimized conditions, the concentration of chlorogenic acid is the line relation with luminous intensity I in 1～500 μ g/mL scope, and its equation is I=-4.0125x+2047, and wherein x is in μ g/ml, R
2=0.9944.
4.2.2 the precision of method, repeatability and detectability test
Precision: under selected optimum experimental condition, the chlorogenic acid standard solution of 5 μ g/ml is measured for continuous 11 times, mean value is 49.78 μ g/ml, and RSD is 3.62%.
Repeatability: 50 μ g/ml chlorogenic acid standard solution are divided into 6 parts, measure respectively, mean value is 50.05 μ g/ml, and RSD is 3.46%.
Detectability: 1.0 μ g/ml
4.3 interference test
Chlorogenic acid derives from blade, other components of coexistence in leaching process, and they possibly measure chlorogenic acid to this chemical luminous system has certain influence.For this reason, studied 100 times Na
+, Cl
-, K
+, Br
-, CO
3 2-, SO
4 2-And NO
3 -50 times Fe
3+, Ca
2+, Cu
2+, Mn
2+10 times starch, dextrin, maltose, sucrose and glucose etc. carry out the interference measurement experiment to the chlorogenic acid of 50 μ g/ml respectively.The result shows that they are interference measurement not.
4.4 recovery of standard addition test findings
Table 1 recovery experimental result
Claims (1)
1. method of measuring chlorogenic acid is characterized in that concrete steps are:
(1) preparation reagent: with 1～20 * 10
-3Mol/L H
2SO
4As solvent, preparation 1～20 * 10
-3The l ferrous ammonium sulfate solution of mol/L; As solvent, prepare 1～20 * 10 with redistilled water respectively
-5The methylene blue solution of mol/L, concentration of volume percent are 0.1～5% oxydol, chlorogenic acid standard solution and the sample solution of chlorogenic acid that concentration is 0.001～10mg/mL;
(2) setting of portable injection chemiluminescence detector: l ferrous ammonium sulfate solution that active efflux prepares step (1) respectively and chlorogenic acid standard solution carry out threeway with the flow velocity of 30～60r/min through corresponding pipeline to be mixed, the input injected chemical luminescence analyzer that flows; The methylene blue solution that secondary moving pump prepares step (1) and oxydol inject flow cells with the flow velocity of 50～80r/min through 16 logical introduction valves, annotate 10～20 a seconds time of appearance, and each liquid mixing detects its chemiluminescence intensity I, record data; Above-mentioned test process carries out at 35 ℃ ± 1 ℃ constant temperature;
(3) make typical curve equation: I=-4.0125x+2047, wherein x is in μ g/ml, and concentration is at 1～500 μ g/ml scope, R
2=0.9944;
(4) sample determination of chlorogenic acid: according to step (2) operating process, the chemiluminescence intensity of specimen chlorogenic acid, substitution typical curve equation, calculation sample chlorogenic acid contents: x=(2047-I)/4.0125, wherein x is in μ g/ml.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106383111A (en) * | 2016-10-31 | 2017-02-08 | 内蒙古博奥现代蒙中药技术研究有限公司 | Method for detecting gallic acid in Zhachongshisanwei pills |
| CN109799352A (en) * | 2019-01-29 | 2019-05-24 | 北京健坤禾润科技有限公司 | It chemical illuminating reagent and its is applied in immune detection |
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2012
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Patent Citations (4)
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|---|---|---|---|---|
| JPS6258166A (en) * | 1985-09-09 | 1987-03-13 | Ss Pharmaceut Co Ltd | Method for simultaneous quantitative analysis of water-soluble vitamins in mixed vitamin preparation |
| CN101082587A (en) * | 2007-07-06 | 2007-12-05 | 山东优生医疗科技有限公司 | Method for measuring human body red blood cell acidum folicum content |
| JP2008180723A (en) * | 2008-02-07 | 2008-08-07 | National Institute Of Advanced Industrial & Technology | Apparatus for measuring glucose and ascorbic acid |
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Non-Patent Citations (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106383111A (en) * | 2016-10-31 | 2017-02-08 | 内蒙古博奥现代蒙中药技术研究有限公司 | Method for detecting gallic acid in Zhachongshisanwei pills |
| CN109799352A (en) * | 2019-01-29 | 2019-05-24 | 北京健坤禾润科技有限公司 | It chemical illuminating reagent and its is applied in immune detection |
| CN109799352B (en) * | 2019-01-29 | 2022-03-08 | 北京健坤禾润科技有限公司 | Chemiluminescent reagent and application thereof in immunoassay |
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Application publication date: 20120718 |