CN107367560A - A kind of ultra high efficiency closes the ascorbic method of phase chromatographic determination - Google Patents

A kind of ultra high efficiency closes the ascorbic method of phase chromatographic determination Download PDF

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CN107367560A
CN107367560A CN201710593361.5A CN201710593361A CN107367560A CN 107367560 A CN107367560 A CN 107367560A CN 201710593361 A CN201710593361 A CN 201710593361A CN 107367560 A CN107367560 A CN 107367560A
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ultra
ascorbic
efficiency
closes
phase chromatographic
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董秋月
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Hangzhou Genglan Biotechnology Co Ltd
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Hangzhou Genglan Biotechnology Co Ltd
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N30/06Preparation
    • G01N30/14Preparation by elimination of some components
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N30/06Preparation
    • G01N30/14Preparation by elimination of some components
    • G01N2030/146Preparation by elimination of some components using membranes

Abstract

The invention discloses a kind of ultra high efficiency to close the ascorbic method of phase chromatographic determination, comprises the following steps:5g solid samples are weighed in 50mL test tubes, 10g is added and contains 0.05%H3PO4Methanol aqueous solution after, with high pressure homogenizer homogeneous at twice, be placed in high speed freezing centrifuge and centrifuge, take supernatant 1mL, through filtering with microporous membrane, obtain solid testing sample;5mL fluid samples are weighed in 50mL test tubes, addition contains 0.05%H3PO4Methanol aqueous solution to 20mL, be placed in high speed freezing centrifuge and centrifuge, take supernatant l mL, through filtering with microporous membrane, obtain fluid test sample;It is directly separated using ultra high efficiency conjunction chromatography and determines ascorbic content in testing sample.The present invention quick separating and can detect ascorbic content in testing sample, have efficiently, the advantage such as detection speed is fast, simple to operate, high sensitivity, reproducible, experimental cost is low.

Description

A kind of ultra high efficiency closes the ascorbic method of phase chromatographic determination
Technical field
The present invention relates to mycobacterium tuberculosis detection field, and in particular to a kind of ultra high efficiency closes phase chromatographic determination vitamin C Method.
Background technology
Vitamin C is also known as L-AA, is High Primates animal and the essential nutrients of other a small number of biologies.It is anti-bad Hematic acid can manufacture in most organism by metabolism, but the mankind are most significant exceptions.Most it is widely known by the people Scurvy can be caused by being a lack of vitamin C.In vivo, vitamin C is a kind of antioxidant, and protection body is from free radical Threat, vitamin C simultaneously and a kind of coenzyme.Vitamin C is a kind of indispensable material for maintaining health of human body.It is The catalyst of living cells redox reaction, a variety of metabolism in vivo are participated in, have and promote internal a variety of physiology rapidly synthesized to make With.Scurvy easily occurs for the C that is deficient in vitamin in body, can be caused death when serious.Medical research also found that it not only has life Reason is active, and the formation of nitrosamine can be prevented in human body, has certain protective effect on cancer risk, increase body is to infectious disease Resistance, it is effective in cure prosperous to hepatitis, cirrhosis.It is again simultaneously a kind of preferable food antioxidant.But human body in itself can not Synthesis, it is necessary to supplied by meals, vitamin C is widely present in fresh water fruits and vegetables.Therefore Accurate Determining dimension life is established The method of plain C content has very important realistic meaning.
The method of vitamin E and C typically has iodimetric titration, colorimetric method, potentiometry, ultraviolet spectrophotometry, fluorescence method etc., These method choices are poor, easily disturbed by compositions such as other reproducibility composition, pyruvic acid and sugar, required reagent is more, It is cumbersome.High performance liquid chromatography is quickly grown in recent years, it have the characteristics that it is efficient, quick, but in experimentation Organic solvent consuming is more, and experimental cost is higher, and certain pollution is more or less caused to environment.Numerous documents are traditional Separation analysis is carried out to vitamin C in reversed-phase liquid chromatography, causes retention time because vitamin C has high polarity in itself It is shorter, seriously affect ascorbic accurate quantitative analysis.The method detected using ultra high efficiency conjunction phase chromatography to vitamin C But be rarely reported, ultra high efficiency close phase chromatogram in addition to advantage possessed by conventional ultra high effect liquid phase chromatogram, also with it is overcritical Fluid Chromatography technology is combined, with supercritical fluid CO2For mobile phase main body, carried out by the solvability of mobile phase Separation, the chromatographic process of analysis.In addition sub-2 μm packings technology, can be by accurately adjusting strength of mobile phase, pressure and temperature Degree, obtains required systemic resolution and selectivity, and the reservation and separation to determinand carry out Effective Regulation, have operation temperature It is low, organic solvent usage amount is few, high sensitivity, it is reproducible, analyze speed is fast the advantages that.Supercritical fluid refers to that material is higher than A kind of states of matter when its critical-temperature and critical pressure, it is neither gas, nor liquid, but it has the low glutinous of gas concurrently The feature of degree, the high density of liquid and the diffusion coefficient between gas-liquid.Vitamin C has at ultraviolet wavelength 245nm Specific absworption peak, sample detect the vitamin C in food after homogeneous, dilution, crossing film, can be qualitative by retention time, peak Area quantitative.Therefore, it is significant to establish a kind of ultra high efficiency conjunction ascorbic method of phase chromatographic determination.
The content of the invention
It is an object of the invention to provide a kind of ultra high efficiency to close the ascorbic method of phase chromatographic determination, and the assay method can Quick separating simultaneously detects ascorbic content in testing sample, have efficiently, detection speed is fast, simple to operate, sensitivity High, the reproducible, advantage such as experimental cost is low.
In order to realize the above-mentioned purpose of the present invention, using following technical scheme:
A kind of ultra high efficiency closes the ascorbic method of phase chromatographic determination, comprises the following steps:
5g solid samples accurately are weighed in 50mL test tubes, are added 10g and are contained 0.05%H3PO4Methanol-water solution after, use High pressure homogenizer homogeneous at twice, is placed in high speed freezing centrifuge and centrifuges, take supernatant 1mL, through filtering with microporous membrane, obtain Solid testing sample;
5mL fluid samples accurately are weighed in 50mL test tubes, and addition contains 0.05%H3PO4Methanol-water solution to 20mL, It is placed in high speed freezing centrifuge and centrifuges, takes supernatant l mL, through filtering with microporous membrane, obtain fluid test sample;
It is directly separated using ultra high efficiency conjunction chromatography and determines the solid testing sample and the fluid test sample In ascorbic content.
Preferably, the solid sample is orange or orange.
Preferably, the fluid sample is water-soluble C100.
Preferably, the high pressure homogenizer twice homogenization condition is:The homogeneous 3min under 14Mpa pressure for the first time, second The secondary homogeneous 5min under 9Mpa pressure.
Preferably, the rotating speed of high speed freezing centrifuge centrifugation be 13000r/min, and temperature is 4 DEG C, and the time is 10min。
Preferably, the aperture of the miillpore filter is less than or equal to 0.22 μm.
Preferably, the analysis condition of the ultra high efficiency conjunction chromatography is:Chromatographic column is Waters Fluoro-Phenyl; Mobile phase is that supercritical carbon dioxide and grade ratio contain 0.05%H3PO4Methanol-water;Flow velocity is 0.8mL/min;Sample size is 1 μL;Column temperature is 30 DEG C;Detection wavelength is 245nm;Dynamic backpressure is 2000psi, using gradient elution, 8% first during 0-0.2min Alcohol, 8%-30% methanol during 3min, 30%-8% methanol during 4min.
Preferably, the specification of the chromatographic column is 3mm × 100mm, 1.7 μm.
Preferably, 0.05%H is contained in the mobile phase3PO4Methanol-water volume ratio be 8:2.
The present invention has the advantages that:
The present invention quick separating and can detect ascorbic content in testing sample, have efficiently, detection speed it is fast, Simple to operate, high sensitivity, the advantage such as reproducible, experimental cost is low.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art and advantage, below will be to implementing The required accompanying drawing used is briefly described in example or description of the prior art, it should be apparent that, drawings in the following description are only Only it is some embodiments of the present invention, for those of ordinary skill in the art, on the premise of not paying creative work, Other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is that the ultra high efficiency of orange in the embodiment of the present invention one closes phase chromatogram;
Fig. 2 is that the ultra high efficiency of orange in the embodiment of the present invention one closes phase chromatogram;
The ultra high efficiency that Fig. 3 is water-soluble C100 in the embodiment of the present invention two closes phase chromatogram.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is The conventional products that can be obtained by commercially available purchase.
Embodiment one
A kind of ultra high efficiency closes ascorbic method in phase chromatographic determination orange, orange
First, standard liquid is prepared
Standard Reserving Solution:0.020g vitamin Cs accurately are weighed, with containing 0.05%H3PO4Methanol-water (8:2, V/V) it is molten Liquid, accurately it is settled in 100mL volumetric flasks, is configured to 200mg/L standard reserving solution, 4 DEG C of refrigerations is stand-by.
Standard working solution:1000,750,500,250,100,50,25 μ L Standard Reserving Solutions of accurate transfer are diluted to respectively 200th, 150,100,50,20,10,5mg/L standard working solution, 4 DEG C of refrigerations are stand-by.
2nd, ultra high efficiency closes phase chromatographic conditions
Chromatographic column is Waters Fluoro-Phenyl (3mm × 100mm, 1.7 μm);Mobile phase is supercritical carbon dioxide Contain 0.05%H with grade ratio3PO4Methanol-water;Flow velocity is 0.8mL/min;Sample size is 1 μ L;Column temperature is 30 DEG C;Detection wavelength For 245nm;Dynamic backpressure is 2000psi, using gradient elution, 8% methanol during 0-0.2min, and 8%-30% methanol during 3min, 30%-8% methanol during 4min.
3rd, sample treatment
Each 5g of orange, orange is weighed respectively in 50mL test tubes, is added 10g and is contained 0.05%H3PO4Methanol-water (8:2, V/ V) after solution, with high pressure homogenizer homogeneous at twice, the homogeneous 3min under 14Mpa pressure for the first time, second in 9Mpa pressure Lower homogeneous 5min, it is placed in high speed freezing centrifuge and 10min is centrifuged with 13000r/min in 4 DEG C, take supernatant 1mL, via hole diameter is small In 0.22 μm of filtering with microporous membrane, testing sample is obtained.
4th, the selection of flow velocity
When supercritical carbon dioxide is as mobile phase, due to low-viscosity and high diffusion coefficient, separating it During there is higher linear velocity, 3-10 times faster than traditional high performance liquid chromatography of analyze speed, analysis time is shorter, equally, In the case, supercritical fluid chromatography can use longer chromatographic column, obtain bigger post effect and separative efficiency.This implementation Example closes phase chromatogram by using ultra high efficiency, carries out flowing rate to the chromatographic column of sub-2 μm packings, flow velocity is in 0.4-1.0mL/min In the range of optimize, in order to ensure preferable sensitivity, chromatogram column pressure and separation as far as possible with impurity, the present embodiment It is optimum flow rate to select 0.8mL/min.
5th, the selection of modifying agent
Because vitamin C is faintly acid, water soluble vitamin, it can suitably improve the peak of vitamin in acid condition Shape, therefore a small amount of phosphoric acid is added in mobile phase as modifying agent, the present embodiment selection 0.05%H3PO4, 0.1%H3PO4、 0.15%H3PO4As modifying agent, as a result find phosphoric acid-methanol solution of 3 kinds of varying level gradients to ascorbic hangover Peak shape has preferable improvement.In view of mobile phase acidity is higher, chromatographic column can be damaged when using for a long time, reduces post effect, therefore will 0.05%H3PO4Modifying agent as mobile phase.
6th, the selection of dynamic backpressure
One of an important factor for ultra high efficiency is closed in phase chromatogram, and dynamic backpressure is influence separation process.Its main function is control System maintains supercriticality in whole carbon dioxide during whole operation, and when back pressure raises, supercritical fluid densities can increase Greatly, solvability strengthens, post pressure rise.The present embodiment contains 0.05%H with supercritical carbon dioxide and grade ratio3PO4First Alcohol-water is mobile phase, investigates influence of the back pressure to sample separation in the range of 1600~2200psi.As a result show, back pressure is got over Greatly, density, viscosity increase therewith, and post pressure can also raise so that the retention time of object chromatographic peak is reduced.By to sample base Matter, retention time and chromatogram column pressure consider, when back pressure is 2000psi, vitamin C separated with impurity it is best, when Between it is most short, peak shape is symmetrical, in the present embodiment select back pressure be 2000psi.
7th, chromatogram column temperature selects
In order to obtain more preferable separating effect to vitamin C in sample, keep that other chromatographic conditions are constant, and this experiment is also examined Examine influence of the column temperature to separation in the range of 25~50 DEG C.As a result show, with the rise of temperature, ascorbic retention time Gradually extend, this is differed with conventional ultra high effect liquid phase chromatogram.Because chromatogram column temperature is higher, supercritical fluid is close Smaller, solvability reduction is spent, vitamin C may be dissolved for supercritical fluid and exchange capacity decrease so that ascorbic Retention time increases.Furthermore, it is contemplated that when chromatogram column temperature is higher, ascorbic oxidation Decomposition can be accelerated, therefore the present embodiment selects It is optimal separation temperature to select 30 DEG C of temperature.
8th, the range of linearity and sensitivity
Under the conditions of Optimal Experimental, selection 200,150,100,50,20,10,5mg/L vitamin C series standard solution It is measured by above-mentioned chromatographic condition, it is bent with peak area (ordinate y, μ U/s) standard draws sample levels (abscissa x, mg/L) Line, carry out linear regression.As a result show, this method has preferable linear relationship, linear equation y in the range of 5-200mg/L =3.13 × 103X-2.65 × 103, linearly dependent coefficient R2=0.9987.This method is limited to ascorbic detection 1.45mg/kg, quantitatively it is limited to 5mg/kg.
9th, the rate of recovery and precision
Determined according to above-mentioned chromatographic condition sample introduction, calculate its rate of recovery.As a result show, the recovery of standard addition of this method exists Between 96.25%-99.85%, relative standard deviation 0.50%-0.72%, the rate of recovery and repeatability are preferable.
Tenth, actual sample determines
Using above-mentioned optimum experimental condition, each 5g of orange, orange is weighed respectively in 50mL test tubes, is added 10g and is contained 0.05%H3PO4Methanol-water (8:2, V/V) after solution, with high pressure homogenizer homogeneous at twice, for the first time under 14Mpa pressure Homogeneous 3min, second of homogeneous 5min under 9Mpa pressure, is placed in high speed freezing centrifuge and is centrifuged in 4 DEG C with 13000r/min 10min, supernatant 1mL is taken, via hole diameter is less than 0.22 μm of filtering with microporous membrane, is surveyed according to above-mentioned chromatographic condition direct injected It is fixed, see Fig. 1 and Fig. 2, the results showed that, Vitamin C content is 184.35mg/kg in orange, and Vitamin C content is in orange 2163.62mg/kg.This method need not carry out complicated pre-treatment, direct injected, directly detect, process is simple, analysis time is short, Reliable results, loss of the vitamin C for a long time, in complicated, cumbersome preceding processing and detection process is effectively avoided, largely The accuracy for improving vitamin C in orange and detecting.
Embodiment two
A kind of ultra high efficiency closes ascorbic method in the water-soluble C of phase chromatographic determination
First, standard liquid is prepared
Standard Reserving Solution:0.020g vitamin Cs accurately are weighed, with containing 0.05%H3PO4Methanol-water (8:2, V/V) it is molten Liquid, accurately it is settled in 100mL volumetric flasks, is configured to 200mg/L standard reserving solution, 4 DEG C of refrigerations is stand-by.
Standard working solution:1000,750,500,250,100,50,25 μ L Standard Reserving Solutions of accurate transfer are diluted to respectively 200th, 150,100,50,20,10,5mg/L standard working solution, 4 DEG C of refrigerations are stand-by.
2nd, ultra high efficiency closes phase chromatographic conditions
Chromatographic column is Waters Fluoro-Phenyl (3mm × 100mm, 1.7 μm);Mobile phase is supercritical carbon dioxide With grade ratio the methanol-water containing 0.05%H3PO4;Flow velocity is 0.8mL/min;Sample size is 1 μ L;Column temperature is 30 DEG C;Detect ripple A length of 245nm;Dynamic backpressure is 2000psi, using gradient elution, 8% methanol during 0-0.2min, and 8%-30% first during 3min Alcohol, 30%-8% methanol during 4min.
3rd, sample treatment
5mL fluid samples accurately are weighed in 50mL test tubes, and addition contains 0.05%H3PO4Methanol-water solution to 20mL, It is placed in high speed freezing centrifuge and centrifuges, takes supernatant l mL, the filtering with microporous membrane that 0.22 μm of via hole diameter, obtain treating test sample Product.
4th, the selection of flow velocity
When supercritical carbon dioxide is as mobile phase, due to low-viscosity and high diffusion coefficient, separating it During there is higher linear velocity, 3-10 times faster than traditional high performance liquid chromatography of analyze speed, analysis time is shorter, equally, In the case, supercritical fluid chromatography can use longer chromatographic column, obtain bigger post effect and separative efficiency.This implementation Example closes phase chromatogram by using ultra high efficiency, carries out flowing rate to the chromatographic column of sub-2 μm packings, flow velocity is in 0.4-1.0mL/min In the range of optimize, in order to ensure preferable sensitivity, chromatogram column pressure and separation as far as possible with impurity, the present embodiment It is optimum flow rate to select 0.8mL/min.
5th, the selection of modifying agent
Because vitamin C is faintly acid, water soluble vitamin, it can suitably improve the peak of vitamin in acid condition Shape, therefore a small amount of phosphoric acid is added in mobile phase as modifying agent, the present embodiment selection 0.05%H3PO4, 0.1%H3PO4、 0.15%H3PO4As modifying agent, as a result find phosphoric acid-methanol solution of 3 kinds of varying level gradients to ascorbic hangover Peak shape has preferable improvement.In view of mobile phase acidity is higher, chromatographic column can be damaged when using for a long time, reduces post effect, therefore will 0.05%H3PO4Modifying agent as mobile phase.
6th, the selection of dynamic backpressure
One of an important factor for ultra high efficiency is closed in phase chromatogram, and dynamic backpressure is influence separation process.Its main function is control System maintains supercriticality in whole carbon dioxide during whole operation, and when back pressure raises, supercritical fluid densities can increase Greatly, solvability strengthens, post pressure rise.The present embodiment contains 0.05%H with supercritical carbon dioxide and grade ratio3PO4First Alcohol-water is mobile phase, investigates influence of the back pressure to sample separation in the range of 1600~2200psi.As a result show, back pressure is got over Greatly, density, viscosity increase therewith, and post pressure can also raise so that the retention time of object chromatographic peak is reduced.By to sample base Matter, retention time and chromatogram column pressure consider, when back pressure is 2000psi, vitamin C separated with impurity it is best, when Between it is most short, peak shape is symmetrical, in the present embodiment select back pressure be 2000psi.
7th, chromatogram column temperature selects
In order to obtain more preferable separating effect to vitamin C in sample, keep that other chromatographic conditions are constant, and this experiment is also examined Examine influence of the column temperature to separation in the range of 25~50 DEG C.As a result show, with the rise of temperature, ascorbic retention time Gradually extend, this is differed with conventional ultra high effect liquid phase chromatogram.Because chromatogram column temperature is higher, supercritical fluid is close Smaller, solvability reduction is spent, vitamin C may be dissolved for supercritical fluid and exchange capacity decrease so that ascorbic Retention time increases.Furthermore, it is contemplated that when chromatogram column temperature is higher, ascorbic oxidation Decomposition can be accelerated, therefore the present embodiment selects It is optimal separation temperature to select 30 DEG C of temperature.
8th, the range of linearity and sensitivity
Under the conditions of Optimal Experimental, selection 200,150,100,50,20,10,5mg/L vitamin C series standard solution It is measured by above-mentioned chromatographic condition, it is bent with peak area (ordinate y, μ U/s) standard draws sample levels (abscissa x, mg/L) Line, carry out linear regression.As a result show, this method has preferable linear relationship, linear equation y in the range of 5-200mg/L =3.13 × 103X-2.66 × 103, linearly dependent coefficient R2=0.9989.This method is limited to 1.5mg/ to ascorbic detection Kg, quantitatively it is limited to 5mg/kg.
9th, the rate of recovery and precision
Determined according to above-mentioned chromatographic condition sample introduction, calculate its rate of recovery.As a result show, the recovery of standard addition of this method exists Between 96.05%-101.15%, relative standard deviation 0.52%-0.76%, the rate of recovery and repeatability are preferable.
Tenth, actual sample determines
Using above-mentioned optimum experimental condition, the water-soluble C100 of 5mL are accurately weighed in 50mL test tubes, and addition contains 0.05%H3PO4 Methanol-water solution to 20mL, be placed in high speed freezing centrifuge and centrifuge, take supernatant l mL, the micropore that 0.22 μm of via hole diameter Membrane filtration, determined according to above-mentioned chromatographic condition direct injected, see Fig. 3, the results showed that, Vitamin C content is in orange 324.78mg/L.This method need not carry out complicated pre-treatment, direct injected, directly detect, and process is simple, analysis time is short, knot Fruit is reliable, effectively avoids loss of the vitamin C for a long time, in complicated, cumbersome preceding processing and detection process, significantly Improve the accuracy that vitamin C detects in orange.
The present invention closes phase chromatogram using ultra high efficiency and vitamin C is detected, and whole pretreatment process is simple, analysis time Short, reliable results, loss of the vitamin C for a long time, in complicated, cumbersome preceding processing and detection process is effectively avoided, very greatly The accuracy for improving food vitamins C detections of degree.Because mobile phase is carbon dioxide, supercritical fluid, to environment Hardly pollute, operating cost is low.
Described above is only the preferred embodiments of the present invention, based on the embodiment in the present invention, ordinary skill people The every other embodiment that member is obtained under the premise of creative work is not made, it should all belong to the model that the present invention protects Enclose.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, may be used also To make some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.

Claims (9)

1. a kind of ultra high efficiency closes the ascorbic method of phase chromatographic determination, it is characterised in that comprises the following steps:
5g solid samples accurately are weighed in 50mL test tubes, are added 10g and are contained 0.05%H3PO4Methanol-water solution after, use high pressure Homogenizer homogeneous at twice, is placed in high speed freezing centrifuge and centrifuges, take supernatant 1mL, through filtering with microporous membrane, obtain solid Testing sample;
5mL fluid samples accurately are weighed in 50mL test tubes, and addition contains 0.05%H3PO4Methanol-water solution to 20mL, be placed in Centrifuged in high speed freezing centrifuge, take supernatant l mL, through filtering with microporous membrane, obtain fluid test sample;
It is directly separated and determines in the solid testing sample and the fluid test sample using ultra high efficiency conjunction chromatography and ties up Raw plain C content.
2. close the ascorbic method of phase chromatographic determination according to the ultra high efficiency described in claim 1, it is characterised in that the solid-like Product are orange or orange.
3. ultra high efficiency according to claim 1 closes the ascorbic method of phase chromatographic determination, it is characterised in that the liquid Sample is water-soluble C100.
4. ultra high efficiency according to claim 1 closes the ascorbic method of phase chromatographic determination, it is characterised in that the high pressure Homogenizer twice homogenization condition is:The homogeneous 3min under 14Mpa pressure for the first time, second of homogeneous 5min under 9Mpa pressure.
5. ultra high efficiency according to claim 1 closes the ascorbic method of phase chromatographic determination, it is characterised in that the high speed The rotating speed of refrigerated centrifuge centrifugation is 13000r/min, and temperature is 4 DEG C, time 10min.
6. ultra high efficiency according to claim 1 closes the ascorbic method of phase chromatographic determination, it is characterised in that the micropore The aperture of filter membrane is less than or equal to 0.22 μm.
7. ultra high efficiency according to claim 1 closes the ascorbic method of phase chromatographic determination, it is characterised in that the superelevation Effect close chromatography analysis condition be:Chromatographic column is Waters Fluoro-Phenyl;Mobile phase is supercritical carbon dioxide Contain 0.05%H with grade ratio3PO4Methanol-water;Flow velocity is 0.8mL/min;Sample size is 1 μ L;Column temperature is 30 DEG C;Detection wavelength For 245nm;Dynamic backpressure is 2000psi, using gradient elution, 8% methanol during 0-0.2min, and 8%-30% methanol during 3min, 30%-8% methanol during 4min.
8. ultra high efficiency according to claim 7 closes the ascorbic method of phase chromatographic determination, it is characterised in that the chromatogram The specification of post is 3mm × 100mm, 1.7 μm.
9. ultra high efficiency according to claim 7 closes the ascorbic method of phase chromatographic determination, it is characterised in that the flowing Contain 0.05%H in phase3PO4Methanol-water volume ratio be 8:2.
CN201710593361.5A 2017-07-20 2017-07-20 A kind of ultra high efficiency closes the ascorbic method of phase chromatographic determination Pending CN107367560A (en)

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CN106814155A (en) * 2017-03-10 2017-06-09 国家烟草质量监督检验中心 The chiral analysis of nicotine close phase chromatographic tandem mass spectrography in a kind of tomato
CN106814155B (en) * 2017-03-10 2019-05-17 国家烟草质量监督检验中心 The chiral analysis of nicotine closes phase chromatographic tandem mass spectrography in a kind of tomato

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