CN105510474A - Method for measuring organic acid and soluble sugar in fruit or fruit wine at same time - Google Patents
Method for measuring organic acid and soluble sugar in fruit or fruit wine at same time Download PDFInfo
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- G01—MEASURING; TESTING
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Abstract
The invention provides a method for measuring organic acid and soluble sugar in fruit or fruit wine at the same time. A high performance liquid chromatograph, a diode array detector or a DAD and a differential detector are connected in series for use, the ultraviolet wavelength is 190-280 nm, an analysis column is a hydrogen ion exchange column, a flowing phase is a water phase A, namely, a sulfuric acid solution with the concentration of 0.001 mol/L-0.005 mol/L, organic acid B is acetonitrile, the volume percent of A is 96-100%, the volume percent of B is 0-4%, the flowing speed is 0.3-0.5 mL/min, the sample size is 5-20 microliters, the detection time is 55 min, and the content of 12 types of organic acid and 3 types of sugar in fruit are quantitatively analyzed and calculated through an external standard method. The method has the advantages that the component peak presentation property is good, the resolution ratio is high, repeatability is good, and the composition and structure characteristics of various types of organic acid and sugar can be represented.
Description
Technical field
The invention belongs to the detection field in fruit or fruit wine, be specifically related to the method for organic acid in a kind of Simultaneously test fruit or fruit wine and soluble sugar.
Background technology
Soluble sugar in fruit and organic acid content are the important foundations that fruit quality is formed, closely related with its flavor quality, constantly change in the biological development process of fruit, have close relationship with the metabolism of saccharic acid in fruit and other materials, running, synthesis and conversion etc.The kind of fruit saccharic acid, content and dynamic change thereof are also one of study hotspots of producing of current high-quality.Correlative study in recent years on the main fruit trees such as oranges and tangerines, apple, Lee, apricot has been reported.
Organic acid assay method has the chromatography of ions, capillary electrophoresis, vapor-phase chromatography etc.The chromatography of ions generally does leacheate with rare strong acid, can cause certain corrosion to instrument; Capillary electrophoresis degree of separation is higher, but repeatability is bad, and precision is not high, and the organic acid content surveyed is inaccurate; Vapor-phase chromatography needs to carry out derivatization treatment to the organic acid of difficulty volatilization, and step is lengthy and tedious.High performance liquid chromatography is the mensuration organic acid method generally adopted at present, and the method is simple, convenient, precision is high, overcomes the deficiency of above method.Because sugar does not absorb in Uv and visible light district, when therefore adopting high effective liquid chromatography for measuring sugar, in order to improve sensitivity for analysis, need detect with UV or DAD after proper method is derivative.
Differential refraction detector (RefractiveIndexDetector, RID) is a kind of highly stable and sensitive liquid chromatography and gel chromatography detecting device.Its principle is the sample peak that change by comparing index of refraction detects mobile phase.For the material only having ultraviolet end to absorb, relatively low with UV detect sensitivity, easily there is baseline wander simultaneously, and ideal result can be obtained with differential refraction detector, as polymkeric substance, sugar, organic acid etc.Differential pulse polarograpll method (RID) analyzes sugar, and sample does not need to derive, and method is quick, direct, but RID cannot carry out gradient elution, and detection sensitivity is low.Versatility quality concentration detector (ELSD) all can produce response to not having the material of uv absorption, fluorescence or electroactive material and generation end uv absorption, is suitable for low content carbohydrate and measures.As Yan Jianrong pulls the eight kinds of sugar such as fructose, glucose, mannose adopted in ELSD2000 evaporative light-scattering detector-high-performance liquid chromatogram determination cordate houttuynia.
In prior art, great majority are the mensuration utilizing different chromatographic columns and pre-treatment to carry out sugar and acid respectively, also do not occur adopting high performance liquid chromatography DAD detecting device to connect the pertinent literature of 12 organic acids (oxalic acid, citric acid, tartrate, chinic acid, succinic acid, malic acid, fumaric acid, acetic acid, propionic acid, isopropyl acid, butyric acid, benzoic acid) in Composition distribution Simultaneously test fruit and 3 kinds of sugar (fructose, sucrose, glucose) or report.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, the method for organic acid in a kind of Simultaneously test fruit or fruit wine and soluble sugar is provided.The present invention adopts 12 kinds of organic acids (being respectively: oxalic acid, citric acid, tartrate, chinic acid, succinic acid, malic acid, fumaric acid, acetic acid, propionic acid, isopropyl acid, butyric acid, benzoic acid) and 3 kinds of sugar (fructose, sucrose, glucose) in high performance liquid chromatography DAD detecting device-series connection Composition distribution energy Simultaneously test fruit, simple and quick.
For achieving the above object, the present invention adopts following technical scheme:
Organic acid in Simultaneously test fruit or fruit wine and a method for soluble sugar, comprise the steps:
(1) preparation of standard model solution
Take chromatographically pure oxalic acid, citric acid, tartrate, succinic acid, malic acid, chinic acid, fumaric acid, acetic acid, propionic acid, isobutyric acid, butyric acid, benzoic acid (12 kinds of organic acids) respectively, glucose, fructose, sucrose (3 kinds of sugar), so can prepare these 12 kinds of organic acid standard items variable concentrations gradients in the standard model solution of 0.01-1mg/mL, 3 kinds of saccharic composition variable concentrations gradients at the standard model solution of 0.1-75mg/mL with mobile phase aqueous phase;
(2) preparation of liquid to be measured
The preparation of fresh sample: take the fresh sample of 10g and be placed in tissue mashing machine, and add 100ml80wt% ethanolic solution homogenate 1min, get the homogenate of 10ml to separate supernatant after the centrifugal 10min of 3000rpm/min, supernatant is proceeded in 50mL volumetric flask, and centrifugal residue washes twice with 80wt% ethanolic solution again, each 15ml, centrifugal 10min; Merge supernatant, mix to obtain extract with 80wt% ethanolic solution constant volume; Get 5.00mL extract in evaporating dish, and at 70 DEG C water bath with thermostatic control ethanol evaporation, evaporation obtain residue redistilled water constant volume mix to obtain sample liquid to 10mL, this sample liquid is crossed 0.45 μm of filter membrane and obtains liquid to be measured;
The preparation of dry sample: claim 2 ~ 3g dry sample and ground 60 orders, then with 80wt% ethanolic solution for solvent, water bath with thermostatic control lixiviate 30min at 80 DEG C, and collected by centrifugation extract, then by extract evaporate to dryness, the material that evaporate to dryness obtains adopts 2ml ultrapure water to dissolve to obtain sample liquid, this sample liquid is crossed 0.45 μm of filter membrane and namely obtains liquid to be measured;
(3) use the HPLC instrument comprising quaternary pump, column oven, DAD detecting device/Composition distribution and workstation and form, on hydrogen ion exchange column, with acetonitrile-sulfuric acid solution for mobile phase isocratic elution is separated various organic acid, chromatographic condition is as follows:
Analytical column: hydrogen ion exchange column;
Mobile phase: the sulfuric acid solution of aqueous phase A:0.001mol/L-0.005mol/L; Organic phase B: acetonitrile; And the percent by volume of A is that the percent by volume of 100-96%, B is for being 0-4%;
Flow velocity: 0.3-0.5mL/min;
Type of elution: isocratic elution;
Determined wavelength: 190nm-280nm;
Temperature: 25-50 DEG C;
Sampling volume: 5-20 μ L;
Detection time: 55min;
(4) measure: get standard model solution prepared by step (1) and liquid to be measured prepared by step (2) respectively, adopt the chromatographic condition of step (3) to carry out sample detection, adopt 10 kinds of organic acids in quantified by external standard method analytical calculation fruit and 3 kinds of sugared content afterwards.
Hydrogen ion exchange column described in step (3) is RezexROA-OrganicAcidH+NewColumn250 × 4.6mm.
Described fruit comprises the one in loquat, grape, fragrant mamoncillo, the fruit of Chinese magnoliavine, oranges and tangerines; Described fruit wine is grape wine.
beneficial effect of the present invention is:
The invention provides the high performance liquid chromatography of 12 kinds of organic acids and 3 kinds of sugared component contents in a kind of Simultaneously test fruit or fruit wine, there is simple and quick feature.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of standard model solution of the present invention; 1-oxalic acid, 2-citric acid, 3-tartrate, 4-malic acid, 5-succinic acid, 6-fumaric acid, 7-acetic acid, 8-chinic acid, 9-propionic acid, 10-isobutyric acid, 11-butyric acid;
Fig. 2 is Composition distribution of the present invention sugar component standard items figure; 13-glucose, 14-sucrose, 15-fructose;
Fig. 3 is the high-efficient liquid phase chromatogram of Murcott young fruit pulp.
Embodiment
The present invention's the following example further illustrates the present invention, but protection scope of the present invention is not limited to the following example.
The typical curve regression equation of table 112 kind of organic acid, soluble sugar, related coefficient, linear lower limit
embodiment 1
1. the organic acid in grape fruit wine and oranges and tangerines, loquat fruit and soluble sugar measure
1 materials and methods
1.1 materials and reagent
The organic acid available from Sigma such as oxalic acid, citric acid, tartrate, malic acid, succinic acid, fumaric acid, acetic acid, propionic acid, isobutyric acid, butyric acid.
1.2 instrument and equipment
L-2000 type high performance liquid chromatograph (HIT), is furnished with diode array detector (DAD), quaternary pump, column oven, automatic sampler etc.; SartoriusBS224S analytical balance (German Sai Duolisi); FW100 high speed Universalpulverizer (Tianjin Stettlen Instrument Ltd.); ALLEGRATM64R high speed freezing centrifuge (BECKMAN company of the U.S.).
1.3 test method
1.3.1 the drafting of typical curve
Accurately take the titer that standard model 80wt% ethanol is mixed with 1mg/mL, be made into the mixed sample of variable concentrations by stepwise dilution method ,-20 DEG C save backup;
1.3.2 sample pre-treatments
2g freeze-dried fruit sample → 20mL80wt% ethanol grinding → 80 DEG C of centrifugal 10min of water-bath 30min lixiviate → 3000rpm/min separate supernatant → repetition four times → collect extract → evaporate to dryness → 2mL ultrapure water and dissolve, sample liquid is crossed 0.45 μm of filter membrane, for liquid-phase chromatographic analysis;
100mL fruit wine sample (wine) reduced pressure concentration → freeze-drying → 10mL80wt% ethanol dissolving → constant volume → sample liquid is crossed 0.45 μm of filter membrane, for liquid-phase chromatographic analysis;
1.3.3 chromatographic condition
Analytical column: RezexROA-OrganicAcidH+NewColumn (300 × 7.8mm, 8 μm), mobile phase: A:0.002mol/L sulfuric acid; B: acetonitrile; A:B:96:4; Flow velocity: 0.4mL/min; Type of elution: isocratic elution; Detect: 210nm-280nm; Temperature: 25 DEG C; Sampling volume: 10 μ L; Detection time: 30min;
2 results and analysis
The selection of 2.1 chromatographic conditions
Through experiment screening, show that the Optimum separation condition of 10 kinds of organic acids on RezexROA-OrganicAcidH+NewColumn post common in fruit is: 0.002mol/L sulfuric acid-acetonitrile, 96:4 is mobile phase, flow velocity 0.4mLmin
-1, sample size is 10 μ L.Under the chromatographic condition determined, enter 10 kinds of organic acid mixed standard solutions, draw the retention time at each peak; Then get 10 organic acid list mark solution respectively, sample introduction under similarity condition, draws various organic acid retention time, thus determines the organic acid that in hybrid standard chromatogram, each peak is corresponding.Oxalic acid (10.17min), citric acid (12.43min), tartrate (13.00min), malic acid (14.77min), succinic acid (18.45min), fumaric acid (20.77min), acetic acid (22.91min), propionic acid (26.31min), isobutyric acid (29.09min), butyric acid (31.37min) etc. are followed successively by hybrid standard chromatogram.
In 2.2 fruits and fruit wine, organic acid measures
Get different fruit samples respectively, according to 1.3.2 processing sample, sample introduction 10 μ L, completes HPLC and measures.Contained by each sample, organic acid the results are shown in Table 2.Organic acid main in grape fruit wine is succinic acid, in pericarp main acid be isobutyric acid.The main acid of Murcott young fruit pulp is citric acid, malic acid and succinic acid.Mainly malic acid in loquat fruit.Different types of organic acid content vinous is different, and as in No. one, Pu, osmanthus, main organic acid is succinic acid, tartrate, malic acid, organic acid main in spine grape wine is tartrate and malic acid.
Organic acid and soluble sugar content (mg/g.DW) in table 2 fruit wine and fruit
embodiment 2
organic acid in the fruit of Chinese magnoliavine, fragrant mamoncillo, golden hesperidium aurantium meat and the mensuration of soluble sugar
1 material and reagent
The organic acid available from Sigma such as oxalic acid, citric acid, tartrate, malic acid, succinic acid, fumaric acid, acetic acid, propionic acid, isobutyric acid, butyric acid;
2 instrument and equipments
L-2000 type high performance liquid chromatograph (HIT), is furnished with diode array detector (DAD), quaternary pump, column oven, automatic sampler etc.; SartoriusBS224S analytical balance (German Sai Duolisi); FW100 high speed Universalpulverizer (Tianjin Stettlen Instrument Ltd.); ALLEGRATM64R high speed freezing centrifuge (BECKMAN company of the U.S.);
3 test methods
The drafting of 3.1 typical curves
Accurately take the titer that standard model 10wt% ethanol is mixed with 1mg/mL, be made into the mixed sample of variable concentrations by stepwise dilution method ,-20 DEG C save backup;
3.2 sample pre-treatments
3g fruit sample → 20mL80wt% ethanol grinding → 80 DEG C of centrifugal 10min of water-bath 30min lixiviate → 3000rpm/min separate supernatant → in triplicate → collection extract → evaporate to dryness → 2ml ultrapure water and dissolve, get part sample liquid and cross 0.45 μm of filter membrane, for liquid-phase chromatographic analysis;
3.3 chromatographic condition
Analytical column: RezexROA-OrganicAcidH+NewColumn (300 × 7.8mm, 8 μm), mobile phase: A:0.005mol/L sulfuric acid; B: acetonitrile; A:B96:4; Flow velocity: 0.33mL/min; Type of elution: isocratic elution; Detect: 210nm-280nm, Composition distribution; Temperature: 30 DEG C; Sampling volume: 10 μ L; Detection time: 55min;
4. result
Organic acid content (mg/g.DW) in table 3 fruit wine and fruit
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (3)
1. the organic acid in Simultaneously test fruit or fruit wine and a method for soluble sugar, is characterized in that: comprise the steps:
(1) preparation of standard model solution
Take chromatographically pure oxalic acid, citric acid, tartrate, succinic acid, malic acid, chinic acid, fumaric acid, acetic acid, propionic acid, isobutyric acid, butyric acid, these 12 kinds of organic acids of benzoic acid respectively, and glucose, fructose, these 3 kinds of sugar of sucrose, then prepare the standard model solution of these 12 kinds of organic acid standard items variable concentrations gradients at 0.01-1mg/mL with mobile phase aqueous phase, and 3 kinds of saccharic composition variable concentrations gradients are at 0.1-
75the standard model solution of mg/mL;
(2) preparation of liquid to be measured
Fresh sample: take the fresh sample of 10g and be placed in tissue mashing machine, and add 100ml80wt% ethanolic solution homogenate 1min, get the homogenate of 10ml to separate supernatant after the centrifugal 10min of 3000rpm/min, supernatant is proceeded in 50mL volumetric flask, and centrifugal residue washes twice with 80wt% ethanolic solution again, each 15ml, centrifugal 10min; Merge supernatant, mix to obtain extract with 80wt% ethanolic solution constant volume; Get 5.00mL extract in evaporating dish, and at 70 DEG C water bath with thermostatic control ethanol evaporation, evaporation obtain residue redistilled water constant volume mix to obtain sample liquid to 10mL, this sample liquid is crossed 0.45 μm of filter membrane and obtains liquid to be measured;
Dry sample: claim 2 ~ 3g dry sample and ground 60 orders, then with 80wt% ethanolic solution for solvent, water bath with thermostatic control lixiviate 30min at 80 DEG C, and collected by centrifugation extract, then by extract evaporate to dryness, the material that evaporate to dryness obtains adopts 2ml ultrapure water to dissolve to obtain sample liquid, this sample liquid is crossed 0.45 μm of filter membrane and namely obtains liquid to be measured;
(3) use the HPLC instrument comprising quaternary pump, column oven, DAD detecting device/Composition distribution and workstation and form, on hydrogen ion exchange column, with acetonitrile-sulfuric acid solution for mobile phase isocratic elution is separated various organic acid, chromatographic condition is as follows:
Analytical column: hydrogen ion exchange column;
Mobile phase: the sulfuric acid solution of aqueous phase A:0.001mol/L-0.005mol/L; Organic phase B: acetonitrile; And the percent by volume of A is that the percent by volume of 100-96%, B is for being 0-4%;
Flow velocity: 0.3-0.5mL/min;
Type of elution: isocratic elution;
Determined wavelength: 190nm-280nm;
Temperature: 25-50 DEG C;
Sampling volume: 5-20 μ L;
Detection time: 55min;
(4) measure: get standard model solution prepared by step (1) and liquid to be measured prepared by step (2) respectively, adopt the chromatographic condition of step (3) to carry out sample detection, adopt 12 kinds of organic acids in quantified by external standard method analytical calculation fruit or fruit wine and 3 kinds of sugared content afterwards.
2. the organic acid in Simultaneously test fruit according to claim 1 or fruit wine and the method for soluble sugar, is characterized in that: the hydrogen ion exchange column described in step (3) is RezexROA-OrganicAcidH+NewColumn250 × 4.6mm.
3. the organic acid in Simultaneously test fruit according to claim 1 or fruit wine and the method for soluble sugar, is characterized in that: described fruit comprises the one in loquat, grape, fragrant mamoncillo, the fruit of Chinese magnoliavine, oranges and tangerines; Described fruit wine is grape wine.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105866310A (en) * | 2016-05-20 | 2016-08-17 | 大连大学 | Method for measuring contents of organic acids in blueberries |
| CN106053637A (en) * | 2016-05-25 | 2016-10-26 | 中国食品发酵工业研究院 | Method for measuring delta 13C values of benzoic acid in milk in online manner |
| CN108398504A (en) * | 2018-03-27 | 2018-08-14 | 石家庄蒎格医药科技有限公司 | A kind of efficient liquid phase method of detection oxalic acid polyethylene glycol naloxone medium-height grass acid content |
| CN108645937A (en) * | 2018-08-09 | 2018-10-12 | 苏州科铭生物技术有限公司 | A kind of propionic acid content assay method based on high performance liquid chromatography |
| CN111122723A (en) * | 2019-01-07 | 2020-05-08 | 中国食品发酵工业研究院有限公司 | Direct determination of citric acid delta in fruit juice13Method of C value |
| CN111198241A (en) * | 2020-03-17 | 2020-05-26 | 乐斯福(明光)有限公司 | Sugar content detection method for baking powder used in cold climate |
| CN112955239A (en) * | 2018-07-27 | 2021-06-11 | 沃特世科技公司 | Liquid chromatography/mass spectrometry method for polar molecule analysis |
| CN113075324A (en) * | 2021-03-30 | 2021-07-06 | 西南大学 | Method for determining sugar and organic acid content in loquat fruits by using HPLC (high Performance liquid chromatography) |
| CN113447585A (en) * | 2021-06-28 | 2021-09-28 | 中国食品药品检定研究院 | Method for evaluating quality of liquid for in-vitro assisted reproduction based on liquid chromatography |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105866310A (en) * | 2016-05-20 | 2016-08-17 | 大连大学 | Method for measuring contents of organic acids in blueberries |
| CN106053637A (en) * | 2016-05-25 | 2016-10-26 | 中国食品发酵工业研究院 | Method for measuring delta 13C values of benzoic acid in milk in online manner |
| CN108398504A (en) * | 2018-03-27 | 2018-08-14 | 石家庄蒎格医药科技有限公司 | A kind of efficient liquid phase method of detection oxalic acid polyethylene glycol naloxone medium-height grass acid content |
| CN112955239A (en) * | 2018-07-27 | 2021-06-11 | 沃特世科技公司 | Liquid chromatography/mass spectrometry method for polar molecule analysis |
| CN112955239B (en) * | 2018-07-27 | 2022-11-29 | 沃特世科技公司 | Liquid chromatography/mass spectrometry method for polar molecule analysis |
| CN108645937A (en) * | 2018-08-09 | 2018-10-12 | 苏州科铭生物技术有限公司 | A kind of propionic acid content assay method based on high performance liquid chromatography |
| CN111122723A (en) * | 2019-01-07 | 2020-05-08 | 中国食品发酵工业研究院有限公司 | Direct determination of citric acid delta in fruit juice13Method of C value |
| CN111198241A (en) * | 2020-03-17 | 2020-05-26 | 乐斯福(明光)有限公司 | Sugar content detection method for baking powder used in cold climate |
| CN113075324A (en) * | 2021-03-30 | 2021-07-06 | 西南大学 | Method for determining sugar and organic acid content in loquat fruits by using HPLC (high Performance liquid chromatography) |
| CN113447585A (en) * | 2021-06-28 | 2021-09-28 | 中国食品药品检定研究院 | Method for evaluating quality of liquid for in-vitro assisted reproduction based on liquid chromatography |
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Application publication date: 20160420 |
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| RJ01 | Rejection of invention patent application after publication |