CN108680683A - Liquid chromatography detects the HPLC methods that carotenoid detects in the mobile phase formula and fruit of carotenoid in fruit - Google Patents
Liquid chromatography detects the HPLC methods that carotenoid detects in the mobile phase formula and fruit of carotenoid in fruit Download PDFInfo
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- 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
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- 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
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- 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
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The present invention relates to chromatography detection fields, specifically, providing the HPLC methods that carotenoid detects in the mobile phase formula and fruit of carotenoid in a kind of liquid chromatography detection fruit.The mobile phase formula includes mobile phase A and Mobile phase B, mobile phase A include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=(80 90):(1‑8):(1‑8):(1‑8)；Mobile phase B include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=(50 60):(5‑15):(15‑20):(15‑20).The mobile phase formula can be used for the measurement of carotenoid content and component in a variety of different fruit samples, be suitable for most C18 chromatographic columns, have the advantages that versatile, sensitive, reproducible for the good separating effect of carotenoid in fruit.
Description
Technical field
The present invention relates to chromatography detection fields, and carotenoid in fruit is detected in particular to a kind of liquid chromatography
Mobile phase formula and fruit in carotenoid detect HPLC methods.
Background technology
Carotenoid is the natural pigment that distributed in nature is most wide, type is most abundant, be widely present in plant, animal and
In algae.Carotenoid typically refers to C40Two class of hydrocarbon (carrotene) and their oxidized derivatives (lutein)
The general name of pigment, they are condensed in structure by 8 isoprene units, typical C40Carotenoid carries ionone
Ring, the hydrogen atom of different location can be replaced by hydroxyl, carbonyl, epoxy group on ring.In plant, carotenoid takes on chloroplaset
Photosynthetic accessory pigments simultaneously protect chlorophyll from strong photo damage, while being also synthesis plant hormone ABA (abscisic
Acid, abscisic acid) precursor.In addition to phytoene and phytofluene, most carotenoid are in yellow, orange
Color or red.Carotenoid there are about 10% is the precursor of vitamin A, be humans and animals food in important component.Fruit
It is the vitals for accumulating carotenoid, the content and composition of carotenoid are the direct embodiments of fruit appearance, nutritional quality,
Analysis detection carotenoid content and component are to evaluate an important indicator of its commodity.
The method of present analysis carotenoid is mainly high performance liquid chromatography (HPLC, High Performance
Liquid Chromatography), this method is sensitive, it is reproducible, be suitable for batch detection, the selection of wherein mobile phase is
Influence the key factor of carotenoid separating effect.However, the mobile phase of most research reports is only applicable to specifically detect sample
Product and chromatographic column, once change testing conditions, are often not achieved ideal separating effect；In addition, due to class Hu in different samples
The difference of radish cellulose content and component, the effect for flowing phase separation are also different.Therefore before new sample starts detection, usually
The component and ratio groping to optimize mobile phase repeatedly are needed, to reach optimal separation effect.
In view of this, special propose the present invention.
Invention content
The mobile phase that the first object of the present invention is to provide carotenoid in a kind of liquid chromatography detection fruit is matched
Side, the mobile phase formula can be used for class Hu trailing plants in a variety of different fruit samples for the good separating effect of carotenoid in fruit
Foretell the measurement of cellulose content and component, be suitable for most C18 chromatographic columns, the multiple types Hu that each component is independent, peak type is good can be obtained
Radish plain color spectrogram and test result have the advantages that versatile, sensitive, reproducible.
The second object of the present invention is to provide a kind of HPLC methods that carotenoid detects in fruit, and this method uses
Above-mentioned mobile phase formula is detected carotenoid in fruit, has carotenoid good separating effect, versatile, clever
Quick, reproducible advantage.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
In a first aspect, the present invention provides the mobile phase formula that a kind of liquid chromatography detects carotenoid in fruit, institute
It includes mobile phase A and Mobile phase B to state formula,
Mobile phase A include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=(80-90):(1-8):
(1-8):(1-8)；
Mobile phase B include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=(50-60):(5-
15):(15-20):(15-20)。
As further preferred technical solution, mobile phase A include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane
Alkane:N-hexane=(82-88):(3-7):(3-7):(3-7)；
Preferably, mobile phase A include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=85:5:
5:5。
As further preferred technical solution, Mobile phase B include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane
Alkane:N-hexane=(52-58):(7-12):(16-19):(16-19)；
Preferably, Mobile phase B include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=55:10:
17.5:17.5。
Second aspect, the present invention provides the HPLC methods that carotenoid in a kind of fruit detects, using above-mentioned liquid phase color
The mobile phase formula of carotenoid is detected carotenoid in fruit in spectrometry detection fruit.
As further preferred technical solution, described includes the steps that HPLC detections, wherein the mobile phase formula
Mixed process is：
(A) contain lycopene in test sample：0min-20min, mobile phase A 25%, Mobile phase B 75%；
(B) without lycopene in test sample：0min-8min, mobile phase A 100%, Mobile phase B 0%；
8min-13min, mobile phase A drop to 0% by 100%, and Mobile phase B increases to 100% by 0%：
13min-20min, mobile phase A 0%, Mobile phase B 100%.
As further preferred technical solution, detector is 2998 type diode array detector of Waters；
Preferably, detection is C18 chromatographic columns with chromatographic column；
Preferably, test sample is dissolved with mobile phase A, and concentration is adjusted to OD450=0.2-0.7；
Preferably, sample size is 10 μ L；
Preferably, flow velocity 0.5-1.5mL/min；
Preferably, column temperature is 20-40 DEG C.
Further include preparing carotenoid test specimens before HPLC detections as further preferred technical solution
The step of product.
As further preferred technical solution, described the step of preparing carotenoid test sample, includes：
(a) sample after crushing is mixed with the mixed liquor of acetone and n-hexane, is filtered, until extracting liquid colourless；
Or, fluid sample is mixed with the mixed liquor of acetone, ethyl acetate and n-hexane, extract, until extract liquor is colourless；
(b) merge the extracting solution or extract liquor in step (a), be concentrated under reduced pressure into dry carotenoid survey concentrate
Test agent.
As further preferred technical solution, it further includes the steps that saponification to be concentrated under reduced pressure into step (b) after drying：
(c) with the dry acetone crude extract of methanol dissolving, 30% isometric methanol-KOH saponification is then added 4 hours with
Go up to obtain saponification liquor；
(d) saponification liquor is concentrated under reduced pressure to minimum volume, adds the mixed liquor of ethyl acetate and n-hexane to be extracted, Zhi Daocui
Take liquid colourless；
(e) merge the extract liquor in step (d), lye when saponification is washed with water, upper layer of extraction liquid is taken to be concentrated under reduced pressure into
The carotenoid test sample that minimum volume must concentrate.
As further preferred technical solution, the volume ratio of acetone and n-hexane is 1 in step (a):1；
Preferably, the volume ratio of acetone, ethyl acetate and n-hexane is 1 in step (a):1:1；
Preferably, the volume ratio of ethyl acetate and n-hexane is 1 in step (d):1.
Compared with prior art, beneficial effects of the present invention are：
The mobile phase of carotenoid is formulated in comprehensive consideration fruit in liquid chromatography detection fruit provided by the invention
The distribution situation of carotenoid selects best ingredient, and the ratio by limiting each ingredient according to the rule of " similar to mix "
Example is so that it is determined that wherein the ratio of polar solvent and nonpolar solvent is reliably divided so that it is guaranteed that the extraction of each pigment is complete
Analyse result.Therefore, above-mentioned mobile phase formula can be used for a variety of different fruits for the good separating effect of carotenoid in fruit
Measurement of carotenoid content and component, including corn, tomato, watermelon, pumpkin, carrot etc. in sample are suitable for most of
C18 chromatographic columns, a variety of carotenoid chromatograms and test result that each component is independent, peak type is good can be obtained, there is versatility
By force, sensitive, reproducible advantage.
The HPLC methods that carotenoid detects in fruit provided by the invention are using above-mentioned mobile phase formula to class in fruit
Carrotene is detected, and has the advantages that carotenoid good separating effect, versatile, sensitive, reproducible.In addition, right
In the sample of no lycopene, by using polar solvent and non-pole in the method adjustment mobile phase of mobile phase formula gradient mixing
The mixed proportion of property solvent, reaches more ideal separating effect；For the sample containing lycopene, match by using mobile phase
The method of the isocratic mixing in side makes appearance time faster, isolates more components.
Description of the drawings
Fig. 1 (a), Fig. 1 (b) and Fig. 1 (c) are respectively corn 1 in embodiment 1, carotenoid in corn 2 and corn 3
HPLC collection of illustrative plates；
Fig. 2 (a) and Fig. 2 (b) are respectively the HPLC collection of illustrative plates of carotenoid in tomato yellow and cyan tomato in embodiment 2；
Fig. 3 (a) is respectively the HPLC for detaching carotenoid in watermelon in embodiment 3 using gradient mobile phase with Fig. 3 (b)
Collection of illustrative plates；
Fig. 4 (a) and Fig. 4 (b) is respectively that the HPLC of carotenoid in isocratic flowing phase separation watermelon is utilized in embodiment 3
Collection of illustrative plates.
Specific implementation mode
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 invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.
As one aspect of the present invention, class is provided in a kind of liquid chromatography detection fruit in some embodiments recklessly
The mobile phase formula of radish element, the formula includes mobile phase A and Mobile phase B,
Mobile phase A include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=(80-90):(1-8):
(1-8):(1-8)；
Mobile phase B include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=(50-60):(5-
15):(15-20):(15-20)。
It is 80 that the volume ratio of each ingredient is typical but non-limiting in mobile phase A:8:5:7、 82:5:8:5、85:5:5:5、
87:8:3:2 or 90:1:5:4 etc..It is 50 that the volume ratio of each ingredient is typical but non-limiting in Mobile phase B:10:20:20、53:
15:15:17、55:10:17.5:17.5、 57:8:18:17 or 60:5:16:19 etc..
The mobile phase of carotenoid is formulated carotenoids in comprehensive consideration fruit in above-mentioned liquid chromatography detection fruit
The distribution situation of element selects best ingredient, and by limiting the ratio of each ingredient to really according to the rule of " similar to mix "
The ratio of fixed wherein polar solvent and nonpolar solvent obtains reliable analysis result so that it is guaranteed that the extraction of each pigment is complete.Cause
This, above-mentioned mobile phase formula can be used for class Hu in a variety of different fruit samples for the good separating effect of carotenoid in fruit
Measurement of radish cellulose content and component, including corn, tomato, watermelon, pumpkin, carrot etc. are suitable for most C18 chromatographies
Each component is independent, peak type is good a variety of carotenoid chromatograms and test result can be obtained in column, have it is versatile, sensitive,
Reproducible advantage.
In a preferred embodiment, mobile phase A include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane
Alkane:N-hexane=(82-88):(3-7):(3-7):(3-7)；
Preferably, mobile phase A include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=85:5:
5:5。
In a preferred embodiment, Mobile phase B include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane
Alkane:N-hexane=(52-58):(7-12):(16-19):(16-19)；
Preferably, Mobile phase B include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=55:10:
17.5:17.5。
By advanced optimizing the volume ratio of each ingredient in mobile phase A and Mobile phase B, mobile phase formula pair is further increased
The completeness of carrotene extraction simultaneously reduces detection error.
As another aspect of the present invention, provide what carotenoid in a kind of fruit detected in some embodiments
HPLC methods, using above-mentioned liquid chromatography detect fruit in carotenoid mobile phase formula to carotenoid in fruit into
Row detection.In above-mentioned fruit carotenoid detect HPLC methods using above-mentioned mobile phase formula to carotenoid in fruit into
Row detection, has the advantages that carotenoid good separating effect, versatile, sensitive, reproducible.
In a preferred embodiment, described includes the steps that HPLC detections, wherein the mobile phase formula is mixed
Conjunction process is：
(A) contain lycopene in test sample：0min-20min, mobile phase A 25%, Mobile phase B 75%；
(B) without lycopene in test sample：0min-8min, mobile phase A 100%, Mobile phase B 0%；
8min-13min, mobile phase A drop to 0% by 100%, and Mobile phase B increases to 100% by 0%：
13min-20min, mobile phase A 0%, Mobile phase B 100%.
For the sample of no lycopene in this preferred embodiment, by using the mixing of mobile phase formula gradient (or
For gradient elution, gradient separations) method adjustment mobile phase in polar solvent and nonpolar solvent mixed proportion, reach more
Ideal separating effect；For the sample containing lycopene, (or washed to be isocratic by using the isocratic mixing of mobile phase formula
De-, isocratic separation) method make appearance time faster, isolate more components.Using above-mentioned preferred embodiment convection current
Dynamic mutually formula is mixed, and can be realized in 20min and be detected to the HPLC of carotenoid in test sample, detection time is short,
It is efficient.
It should be understood that when containing lycopene in test sample, mobile phase formula is carried out according to the process described in (A)
Mixing, i.e., in 0min to 20min, the volume of mobile phase A accounts for the 25% of mobile phase total volume, and the volume of Mobile phase B accounts for stream
The 75% of dynamic phase total volume.
When in test sample without lycopene, the mixing of mobile phase formula is carried out according to the process described in (B), that is, is existed
In 0min to 8min, the volume of mobile phase A accounts for the 100% of mobile phase total volume, no Mobile phase B；In 8min to 13min, stream
The volume of dynamic phase A is gradually decreased to 0% from 100%, and the volume of mobile phase A is gradually increased to 100% from 0%；It is arrived in 13min
In 20min, no mobile phase A, the volume of Mobile phase B accounts for the 100% of mobile phase total volume.
In a preferred embodiment, detector is 2998 type diode array detector of Waters；
Preferably, detection is C18 chromatographic columns with chromatographic column；
Preferably, test sample is dissolved with mobile phase A, and concentration is adjusted to OD450=0.2-0.7；
Preferably, sample size is 10 μ L；
Preferably, flow velocity 0.5-1.5mL/min；
Preferably, column temperature is 20-40 DEG C.
Using the efficient liquid phase detecting system of Waters, detector is 2998 type Diode Array Detectors of Waters for detection
Device.The optional Spherisorb ODS2 chromatographic columns of C18 chromatographic columns, grain size are 5 μm, 250 × 4.6mm of model.Flow velocity is typical but non-
It is restrictive for 0.5mL/min, 0.6mL/min, 0.7mL/min, 0.8mL/min, 0.9mL/min, 1.0mL/min,
1.1mL/min, 1.2mL/min, 1.3mL/min, 1.4mL/min or 1.5mL/min.Typical but non-limiting column temperature is 20
DEG C, 22 DEG C, 24 DEG C, 26 DEG C, 28 DEG C, 30 DEG C, 32 DEG C, 34 DEG C, 36 DEG C, 38 DEG C or 40 DEG C.It is above-mentioned that " concentration is adjusted to OD450=
0.2-0.7 " refers to adjusting liquor capacity after dissolving to make OD450Light absorption value range be in the range of 0.2-0.7, sample concentration
When in the concentration range, the linear relationship of light absorption value and content is good, and quantitative analysis results are more accurate.
This preferred embodiment has advanced optimized the chromatographic conditions such as detector type, chromatographic column type, flow velocity, column temperature,
By the optimization to conditions above, better separating effect is realized, realize the optimization of separating degree and the optimization of peak type.Wherein,
Flow velocity determines the appearance time of sample, and column temperature imitates column and product separation also has certain influence, by flow velocity and column temperature
Setting, to obtain good separating effect.
In a preferred embodiment, further include preparing carotenoid test sample before HPLC detections
The step of.
In a preferred embodiment, described the step of preparing carotenoid test sample, includes：
(a) sample after crushing is mixed with the mixed liquor of acetone and n-hexane, is filtered, until extracting liquid colourless；
Or, fluid sample is mixed with the mixed liquor of acetone, ethyl acetate and n-hexane, extract, until extract liquor is colourless；
(b) merge the extracting solution or extract liquor in step (a), be concentrated under reduced pressure into dry carotenoid survey concentrate
Test agent.
This preferred embodiment provides a kind of preparation process for the carotenoid test sample that need not carry out saponification,
By being filtered or being extracted and concentrated and being dried material sample, the carotenoid test sample concentrated is obtained.
In a preferred embodiment, it further includes the steps that saponification to be concentrated under reduced pressure into step (b) after drying：
(c) with the dry acetone crude extract of methanol dissolving, 30% isometric methanol-KOH saponification is then added 4 hours with
Go up to obtain saponification liquor；
(d) saponification liquor is concentrated under reduced pressure to minimum volume, adds the mixed liquor of ethyl acetate and n-hexane to be extracted, Zhi Daocui
Take liquid colourless；
(e) merge the extract liquor in step (d), lye when saponification is washed with water, upper layer of extraction liquid is taken to be concentrated under reduced pressure into
The carotenoid test sample that minimum volume must concentrate.
This preferred embodiment provides a kind of preparation process for the carotenoid test sample needing to carry out saponification, step
Suddenly " the acetone crude extract " in (c) refers to the extracting solution obtained in step (a) or the extract liquor gained after drying is concentrated under reduced pressure
Substance, the acetone crude extract are further concentrated after saponification and extraction, finally obtain the carotenoid test concentrated
Sample.
Preferably for the carotenoid test sample concentrated, it is transferred into the EP pipes (centrifuge tube) of 1.5mL
In, use N2Remaining solvent is done in air-blowing, and -20 DEG C save backup.
In a preferred embodiment, the volume ratio of acetone and n-hexane is 1 in step (a):1；
Preferably, the volume ratio of acetone, ethyl acetate and n-hexane is 1 in step (a):1:1；
Preferably, the volume ratio of ethyl acetate and n-hexane is 1 in step (d):1.
It is optimized in this preferred embodiment during preparing carotenoid test sample in step (a) and step (d)
The volume ratio of each ingredient, verified in each mixed liquor, and when the volume ratio of each ingredient is the above ratio, effect of extracting is best.
In order to further appreciate that the specific operation process and detection result of the present invention, with reference to specific embodiment to this hair
It is bright to be further described in detail.
Embodiment 1
The HPLC methods that carotenoid detects in corn, include the following steps：
(A) carotenoid test sample is prepared：
(a) corn sample after crushing (is taken the corn sample of three kinds of different cultivars, is denoted as corn 1,2 and of corn respectively
Corn 3) with volume ratio be 1:The mixed liquor mixing of 1 acetone and n-hexane, filters, until extracting liquid colourless；
(b) merge the extracting solution in step (a), be concentrated under reduced pressure into dry acetone crude extract；
(c) with the dry acetone crude extract of methanol dissolving, 30% isometric methanol-KOH saponification is then added 4 hours with
Go up to obtain saponification liquor；
(d) saponification liquor is concentrated under reduced pressure to minimum volume, it is 1 to add volume ratio:1 ethyl acetate and the mixed liquor of n-hexane into
Row extraction, until extract liquor is colourless；
(e) merge the extract liquor in step (d), lye when saponification is washed with water, upper layer of extraction liquid is taken to be concentrated under reduced pressure into
The carotenoid test sample that minimum volume must concentrate；
(B) HPLC is detected：The mixed process of the wherein described mobile phase formula is：
Without lycopene in test sample：0min-8min, mobile phase A 100%, Mobile phase B 0%；
8min-13min, mobile phase A drop to 0% by 100%, and Mobile phase B increases to 100% by 0%：
13min-20min, mobile phase A 0%, Mobile phase B 100%.
Instrument used in embodiment 1 is as follows：The efficient liquid phase detecting system of Waters；Detector is 2998 types of Waters
Diode array detector.
Chromatographic condition：C18 chromatographic columns (Spherisorb ODS2,5 μm, 250 × 4.6mm)；Test sample mobile phase A
Dissolving, concentration are adjusted to OD450=0.2-0.7, with sample introduction after 0.45 μm of membrane filtration, sample size is 10 μ L；Flow velocity is
1.0mL/min；Column temperature is 30 DEG C；
Mobile phase formula is as follows：Mobile phase formula includes mobile phase A and Mobile phase B,
Mobile phase A include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=85:5:5:5；
Mobile phase B include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=55:10:17.5:
17.5。
It is respectively carotenoid in different cultivars corn (corn 1, corn 2 and corn 3) as shown in Fig. 1 (a)-Fig. 1 (c)
HPLC collection of illustrative plates, wherein peak 1 be lutein, peak 2 be zeaxanthin, peak 3 be beta carotene, it can be seen that arrived at the 6th minute
Within 8 minutes, you can separate the similar lutein of structure and zeaxanthin well, and peak type is good.
Embodiment 2
The HPLC methods that carotenoid detects in tomato, detecting step is same as Example 1, the difference is that only institute
It is Vydac 201TP54 chromatographic columns with chromatographic column.
As shown in Fig. 2 (a) and Fig. 2 (b) respectively in tomato yellow and cyan tomato carotenoid HPLC collection of illustrative plates,
Middle peak 1 is lutein, and peak 2 is beta carotene, it can be seen that can be yellow by the leaf in tomato yellow in the 4th minute to 12 minutes
Element and beta carotene are separated, and peak type is good, and beta carotene is not detected in cyan tomato.
Embodiment 3
The HPLC methods that carotenoid detects in watermelon, detecting instrument and chromatographic condition are same as Example 2, difference
Be in and two different separation methods used to same watermelon material respectively in the present embodiment, it is a kind of using isocratic mobile phase into
Row separation, i.e. (A) 0min-20min, mobile phase A 25%, Mobile phase B 75%；Another kind is divided using gradient mobile phase
From, i.e. (B) 0min-8min, mobile phase A 100%, Mobile phase B 0%；8min-13min, mobile phase A are dropped to by 100%
0%, Mobile phase B increases to 100% by 0%：13min-20min, mobile phase A 0%, Mobile phase B 100%.
The HPLC collection of illustrative plates of carotenoid in watermelon is detached using gradient mobile phase as Fig. 3 (a) and Fig. 3 (b) are shown,
Middle peak 1 is lycopene, and peak 2 is beta carotene, it can be seen that can be by lycopene and β-Hu in the 6th minute to 12 minutes
Radish element is separated, and peak type is good.
Fig. 4 (a) and Fig. 4 (b) show the HPLC collection of illustrative plates using carotenoid in isocratic flowing phase separation watermelon, wherein
Peak 1 is lycopene, and peak 2 is beta carotene, and peak 3 is phytofluene, and peak 4 is phytoene, it can be seen that
Lycopene and beta carotene can be separated in 4th minute to 8 minutes, faster, peak type is good, and isolates for appearance time
Component it is more, relative to using gradient mobile phase separation for, can be further by phytofluene and phytoene
It separates.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. the mobile phase formula of carotenoid in a kind of liquid chromatography detection fruit, which is characterized in that the formula includes stream
Dynamic phase A and Mobile phase B,
Mobile phase A include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=(80-90):(1-8):(1-
8):(1-8)；
Mobile phase B include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=(50-60):(5-15):
(15-20):(15-20)。
2. the mobile phase formula of carotenoid, feature exist in liquid chromatography detection fruit according to claim 1
In, mobile phase A include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=(82-88):(3-7):(3-
7):(3-7)；
Preferably, mobile phase A include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=85:5:5:5.
3. the mobile phase formula of carotenoid, feature in liquid chromatography detection fruit according to claim 1 or 2
Be, Mobile phase B include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=(52-58):(7-12):
(16-19):(16-19)；
Preferably, Mobile phase B include following volumes than ingredient：Acetonitrile:Methanol:Dichloromethane:N-hexane=55:10:17.5:
17.5。
4. a kind of HPLC methods that carotenoid detects in fruit, which is characterized in that using described in claim any one of 1-3
Liquid chromatography detection fruit in the mobile phase formula of carotenoid carotenoid in fruit is detected.
5. the HPLC methods that carotenoid detects in fruit according to claim 4, which is characterized in that the method packet
The step of including HPLC detections, wherein the mixed process of the mobile phase formula is：
(A) contain lycopene in test sample：0min-20min, mobile phase A 25%, Mobile phase B 75%；
(B) without lycopene in test sample：0min-8min, mobile phase A 100%, Mobile phase B 0%；
8min-13min, mobile phase A drop to 0% by 100%, and Mobile phase B increases to 100% by 0%：
13min-20min, mobile phase A 0%, Mobile phase B 100%.
6. the HPLC methods that carotenoid detects in fruit according to claim 5, which is characterized in that detector is
2998 type diode array detector of Waters；
Preferably, detection is C18 chromatographic columns with chromatographic column；
Preferably, test sample is dissolved with mobile phase A, and concentration is adjusted to OD450=0.2-0.7；
Preferably, sample size is 10 μ L；
Preferably, flow velocity 0.5-1.5mL/min；
Preferably, column temperature is 20-40 DEG C.
7. the HPLC methods that carotenoid detects in fruit according to claim 5 or 6, which is characterized in that described
Further include the steps that preparing carotenoid test sample before HPLC detections.
8. the HPLC methods that carotenoid detects in fruit according to claim 7, which is characterized in that the preparation class
The step of carrotene test sample includes：
(a) sample after crushing is mixed with the mixed liquor of acetone and n-hexane, is filtered, until extracting liquid colourless；
Or, fluid sample is mixed with the mixed liquor of acetone, ethyl acetate and n-hexane, extract, until extract liquor is colourless；
(b) merge the extracting solution or extract liquor in step (a), be concentrated under reduced pressure into dry the carotenoid test specimens concentrated
Product.
9. the HPLC methods that carotenoid detects in fruit according to claim 8, which is characterized in that subtract in step (b)
Pressure further includes the steps that saponification after being concentrated to dryness：
(c) with the dry acetone crude extract of methanol dissolving, 30% isometric methanol-KOH saponification is then added 4 hours or more must
Saponification liquor；
(d) saponification liquor is concentrated under reduced pressure to minimum volume, adds the mixed liquor of ethyl acetate and n-hexane to be extracted, until extract liquor
It is colourless；
(e) merge the extract liquor in step (d), lye when saponification is washed with water, upper layer of extraction liquid is taken to be concentrated under reduced pressure into minimum
The carotenoid test sample that volume must concentrate.
10. the HPLC methods that carotenoid detects in fruit according to claim 9, which is characterized in that in step (a)
The volume ratio of acetone and n-hexane is 1:1；
Preferably, the volume ratio of acetone, ethyl acetate and n-hexane is 1 in step (a):1:1；
Preferably, the volume ratio of ethyl acetate and n-hexane is 1 in step (d):1.
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