CN106290637B - A kind of polycyclic aromatic hydrocarbon enters the method for inspection of cabbage heart main path - Google Patents
A kind of polycyclic aromatic hydrocarbon enters the method for inspection of cabbage heart main path Download PDFInfo
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Abstract
The invention discloses the methods of inspection that a kind of polycyclic aromatic hydrocarbon enters cabbage heart main path, comprising the following steps: (1) cabbage heart seedling is colonized in culture substrate, is at least divided into blade validation group, root validation group；(2) the polycyclic aromatic hydrocarbon mixed solution of equal weight and concentration is added into the Brassica campestris leaves of blade validation group and the cabbage heart culture substrate of root validation group respectively, grows to harvest time to cabbage heart, harvests；(3) cabbage heart of harvest is rinsed with water completely, is dry, pulverize；(4) it extracts and obtains polycyclic aromatic hydrocarbon extracting solution；(5) with the content of polycyclic aromatic hydrocarbon in high performance liquid chromatograph detection polycyclic aromatic hydrocarbon extracting solution, the content of polycyclic aromatic hydrocarbon in blade validation group and root validation group is compared.Method of the invention is simple to operation, can find the main path that polycyclic aromatic hydrocarbon enters cabbage heart.
Description
Technical field
The present invention relates to the methods of inspection that vegetables inspection field more particularly to polycyclic aromatic hydrocarbon enter cabbage heart main path.
Background technique
Cabbage heart is also known as tender flower stalk, is the annual herb plant of Cruciferae Brassica genus, is the vegetables that Chinese often eat
One of, it is characterized in stem's green, there is tiny dish leaf, blade is bottle green, and has the little Hua of yellow between blade, is one independent
Kind.Cabbage heart is through Long-term selection, and to form different kinds by the easy bolting material of Chinese cabbage.Cabbage heart is Chinese wide
The special vegetable in east, quality is tender, flavor is palatable, and energy annually cultivating, so be continental vegetables on Guangdong, Guangxi and other places,
The ground such as anniversary transportation and sales Hong Kong, Macao, become the main vegetables of outlet.There are also a small amount of to be exported to America and Europe, is considered as rare vegetables, is praised
For " after in dish " and " hats of vegetable product ".Cabbage heart unique flavor, the tender taste of matter is good, convenient, various in style, wide adaptability, raw
It is long rapid, it occupies an important position in vegetables year-round supply, especially dull season supply；Cabbage heart both can be cultivated individually, can also
With a kind, interplanting and miscegenation, therefore there is biggish cultivated area in the whole nation.After the nineties, with the rapid development of social economy
And a large amount of uses of coal stone oil, cause the carcinogens such as polycyclic aromatic hydrocarbon to enter environment, and endanger the strong of the mankind by food chain
Health.
Polycyclic aromatic hydrocarbons (PAH) (Polycyclic Aromatic Hydrocarbons, PAHs) is also known as polycyclic aromatic compound
Or polycyclic aromatic hydrocarbons, it is a special case of aromatic hydrocarbons, by the virtue for not including heterocycle or substituent group
Fragrant ring is formed.Wherein having much is all known or potential carcinogen, mainly by carbon-containing fuel (firewood, wooden carbon, oil
Rouge) and the imperfect combustion of tobacco etc. caused by.Currently, there are more than 100 kinds in the PAHs of nature discovery, can be generally divided into
Low ring (2~4 ring) PAHs and epipodium (5 rings or more) PAHs, low ring PAHs can be distributed in gaseous environment, but most of
PAHs can only be gathered in inhalable particles surface, especially on the particle less than 5 μm.
PAHs has half volatile and can migrate with atmosphere pollution, falls to soil and plant surface.Agricultural land soil
PAHs accumulation contact scar threatened the quality safeties of crops, therefore, accumulation of the PAHs in vegetables and soil is to people
Class health, which has, directly to endanger.In June, 1998, in the European Environment council of ministers that Denmark holds, the U.S., Canada and Europe
32 states formally propose naphthalene, acenaphthylene, phenanthrene, fluorenes, acenaphthene, anthracene, fluoranthene, pyrene, benzo (a) anthracene, in the wrong, benzo (b) fluoranthene, benzo (k) is glimmering
16 kinds of PAHs column such as anthracene, benzo (a) pyrene, dibenzo (a, h) anthracene, indeno (1,2,3-c, d) pyrene, benzo (g, h, i) perylene
Enter in controlled persistence organic pollutant, U.S.EPA is also included in this 16 kinds of PAHs the pollutant list of priority acccess control
Among.Account for about the 22% of the whole world in the discharge amount of this 16 kinds of PAHs of China, at the forefront in the world, PAHs becomes the soil of Chinese most serious
One of earth pollutant, has reached 28mgkg-1Dry ground.
1961, since Blumer has found PAHs for the first time in the soil, PAHs just caused the extensive pass of people
Note.In recent years, PAHs was classified as one of the important content of environmental monitoring work by most countries.PAHs is a kind of extremely serious
Pollutant, be widely present in soil, atmosphere and water body, have extremely strong carcinogenic, teratogenesis and mutagenesis.Most PAHs
Class compound is difficult to be degraded, and causes seriously to pollute to environment.And it exists in the environment PAHs and is easily absorbed by plants and in vivo
Accumulation, to influence agricultural product security, and can endanger the health of people by food chain.Currently, PAHs is in soil, heavy
Various physics, chemistry and biobehavioral etc. in the environment such as product object, water have caused the very big attention of environmentalist, because
This, the research for entering plant main path to polycyclic aromatic hydrocarbon is extremely necessary.
Summary of the invention
The present invention provides the methods of inspection that a kind of polycyclic aromatic hydrocarbon enters cabbage heart main path, and method is simple to operation, energy
Enough find the main path that polycyclic aromatic hydrocarbon enters cabbage heart.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
A kind of polycyclic aromatic hydrocarbon enters the method for inspection of cabbage heart main path, comprising the following steps:
(1) cabbage heart seedling is colonized in culture substrate, after being colonized 5 ~ 20 days, cabbage heart is at least divided into blade validation group,
Root validation group；
(2) equal weight is added into the Brassica campestris leaves of blade validation group and the cabbage heart culture substrate of root validation group respectively
With the polycyclic aromatic hydrocarbon mixed solution of concentration, continuous processing 1 ~ 6 day, harvest time is grown to cabbage heart, is harvested；
(3) cabbage heart of harvest is rinsed with water completely, after vacuum refrigerating machine is dry, is crushed spare；
(4) smashed cabbage heart organic extraction solvent is impregnated into 15 ~ 48h, then 20 ~ 40min of ultrasonic extraction again, from
The heart collects supernatant, repeats said extracted and operates 1 ~ 2 time, merge supernatant, be concentrated to dryness, with methanol constant volume, with 0.1~0.4
μm aperture membrane filtration obtains polycyclic aromatic hydrocarbon extracting solution；
(5) with the content of polycyclic aromatic hydrocarbon in high performance liquid chromatograph detection polycyclic aromatic hydrocarbon extracting solution, blade validation group is compared
With the content of polycyclic aromatic hydrocarbon in the validation group of root.
Preferably, in the step (2), the concentration of polycyclic aromatic hydrocarbon mixed solution is 100 ~ 900 μ gL-1。
Preferably, in the step (3), the temperature of vacuum freeze drying is -50 ~ -40 DEG C, and pressure is 0.4 ~ 1.0MPa.
Preferably, in the step (4), organic extraction solvent by methylene chloride and n-hexane by volume 2:8 prepare and
At.
Preferably, it in the step (4), is collected after supernatant Florisil silica column.
Preferably, in the step (4), centrifugation uses high speed freezing centrifuge, and revolving speed is 3500 ~ 4000 rpm, temperature
It is 2 ~ 6 DEG C, the time is 8 ~ 20 minutes.
Preferably, in the step (4), the temperature of ultrasonic extraction is 28 ~ 32 DEG C.
Preferably, in the step (4), the temperature of concentration is 35 ~ 40 DEG C.
Preferably, in the step (2), polycyclic aromatic hydrocarbon solution is by naphthalene, acenaphthylene, acenaphthene, fluorenes, phenanthrene, anthracene, fluoranthene, pyrene, benzo
(a) anthracene, bend, benzo (b) fluoranthene, benzo (k) fluoranthene, benzo (a) pyrene, dibenzo (a, h) anthracene, indeno (1,2,3 ,-cd) pyrene and
Benzo (g, h, i) perylene is added acetone solution and is made.
Preferably, in the step (5), chromatographic condition are as follows: chromatographic column is using octadecyl silane as filler, using ladder
Degree elution, mobile phase are the gradient eluent of methanol and pure water composition, and column temperature is 30 DEG C, flow velocity 1ml/min；The gradient is washed
De- program is set as time gradient 0min → 3min → 5min → 30min, and corresponding gradient eluent is by following volumes ratio by flowing
Dynamic phase A methanol and Mobile phase B pure water composition, the percentage by volume gradient 60% → 70% → 80% → 100% of mobile phase A, stream
The percentage by volume gradient 40% → 30% → 20% → 0% of dynamic phase B；Ultraviolet detection wavelength: 254nm.
The invention has the following advantages that
(1) after cabbage heart harvest, using freeze drying process to using rapid draing is carried out, dry speed is accelerated, is dropped
Low dry temperature, it is therefore prevented that polycyclic aromatic hydrocarbon is lost in the drying process, improves the accuracy of inspection；
(2) organic extraction solvent is mixed and made into methylene chloride and n-hexane, preferably mentions polycyclic aromatic hydrocarbon from cabbage heart
It takes and separates, and reduce interference of other effective components to polycyclic aromatic hydrocarbon in cabbage heart, ensure that the precision of inspection；
(3) it extracts, collected after centrifuged supernatant Florisil silica column, there is florisil silica column absorption well to make
With can improve the separating effect of solid phase and liquid phase, be kept completely separate polycyclic aromatic hydrocarbon with cabbage heart powder and come；
(4) during the extraction process, be centrifuged using high speed freezing centrifuge, and extract and concentration control 40 DEG C with
Under, reduce the loss of polycyclic aromatic hydrocarbon during the extraction process, further improves the accuracy of inspection；
(5) content for measuring polycyclic aromatic hydrocarbon by gradient elution using high performance liquid chromatograph, has chosen suitable chromatography
Condition.Preferable separating effect can be obtained, to obtain accurate polycyclic aromatic hydrocarbon content, find the master that polycyclic aromatic hydrocarbon enters cabbage heart
Want approach.
Detailed description of the invention
Fig. 1 is the standard curve of high performance liquid chromatography measurement polycyclic aromatic hydrocarbon.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described, but protection scope of the present invention is not limited to following reality
Apply example.
Test prepares: 16 kinds of PAHs hybrid standard product are purchased from SIGMA-ALORICH company, the U.S., methanol, n-hexane and
Methylene chloride is chromatographically pure.
A kind of polycyclic aromatic hydrocarbon enters the method for inspection of cabbage heart main path, comprising the following steps:
(1) seed is sowed in greenhouse seedlings nursing plate, when the bud of cabbage heart is grown to two leaves wholeheartedly, moves into cultivation bed field planting
Field planting cabbage heart is divided into three groups, respectively blade validation group, root validation group and control group, often after being colonized 14 days by culture
Group at least 6 plants of cabbage hearts；
(2) cabbage heart of blade validation group, ground true leaf part concentration are 600 μ gL-1Polycyclic aromatic hydrocarbon mixed solution is to leaf
Piece progress spraying treatment, continuous processing 3 days；600 μ are added into culture substrate for the cabbage heart of root validation group, subterranean root part
g·L-1Polycyclic aromatic hydrocarbon mixed solution；The cabbage heart of blade validation group and the cabbage heart of root validation group apply the polycyclic of 1.2 μ g every time
Arene solution；The cabbage heart of control group, aerial part and under ground portion are handled with clear water；It is grown to it to harvest time after cultivating 8 days,
Harvest cabbage heart；
(3) cabbage heart of harvest is sufficiently rinsed well with distilled water, the temperature of freeze-drying dry through vacuum freeze drier
Degree is -45 DEG C, and pressure is 0.4 ~ 1.0MPa, and it is spare that drying finishes crushing；
(4) above-mentioned smashed cabbage heart 2g is weighed in centrifuge tube, impregnates 24 h, You Jicui with 30mL organic extraction solvent
Taking solvent by methylene chloride and n-hexane, 2:8 is formulated by volume；Then the cabbage heart after immersion is surpassed in 30 DEG C of water-baths
Sound extracts 30 min, is centrifuged 10 min with high speed freezing centrifuge, temperature is 4 DEG C, and revolving speed is 4000 rpm, by supernatant after centrifugation
It is collected after liquid Florisil silica column, repeats said extracted operation once, merge supernatant, be entirely by the supernatant after merging
It is transferred in rotary evaporation bottle, rotates and be concentrated to dryness under 40 DEG C of constant temperature, with methanol constant volume to 2 mL, cross 0.22 μm of aperture filter membrane
Filtering obtains polycyclic aromatic hydrocarbon extracting solution；
(5) with high performance liquid chromatograph detection polycyclic aromatic hydrocarbon extracting solution in polycyclic aromatic hydrocarbon content, comparison blade validation group,
The content of polycyclic aromatic hydrocarbon in root validation group and control group.
More specifically, in step (1), when field planting is to move into water planting cultivation bed to cultivate, and water planting is using deep-flow technique confession
To nutrient solution, and the concentration of its nutrient solution is measured every 5d with conductivity gauge, determine that fertilizer adds according to the variation of conductivity EC value
Enter amount or moisture magnitude of recruitment, EC value is maintained between the mS/cm of 1.8 mS/cm ~ 2.5, pH value is maintained at 6 ~ 6.5.
Water planting carries out in plastics attached-greenhouse, totally 3 cultivation beds, using hole plate seedling growth, when bud is grown to two leaves wholeheartedly
It is colonized in water planting cultivation bed, the seeding row spacing of cabbage heart is 0.2m × 0.2m.
More specifically, in step (2), used polycyclic aromatic hydrocarbon mixed solution preparation process are as follows:
Naphthalene (NAP), luxuriant and rich with fragrance (PHE), fluoranthene (FLT), benzo (a) anthracene (BaA), benzo (a) pyrene (BaP), each are weighed respectively
0.0020g is added n-hexane and is dissolved in the volumetric flask of 1L surely, obtains polycyclic aromatic hydrocarbon stock solution, pipette and dilute polycyclic virtue
600 μ gL are made in hydrocarbon stock solution-1Polycyclic aromatic hydrocarbon mixed solution, wherein 600 μ gL-1Refer to polycyclic aromatic hydrocarbon it is above-mentioned it is a variety of at
Divide melting concn.
Preferably, selection is adopted when high performance liquid chromatography is detected in order to separate target detection thing preferably
It being detected with the mode of gradient elution, determines that gradient elution program is as shown in table 1, column temperature is 30 DEG C, flow velocity 1ml/min,
Ultraviolet detection wavelength: 254nm.
1 polycyclic aromatic hydrocarbon gradient elution program of table
Table1 Gradient elution procedure of PAHs
| Time | Mobile phase A (methanol) | Mobile phase B (pure water) |
| Time/min | Mobile phase A/% | Mobile phase B/% |
| 0 | 60 | 40 |
| 3 | 70 | 30 |
| 5 | 80 | 20 |
| 30 | 100 | 0 |
(5-1) weigh naphthalene (NAP), acenaphthylene (ANY), acenaphthene (ACE), fluorenes (FLU), luxuriant and rich with fragrance (PHE), anthracene (ANT), fluoranthene (FLT),
Pyrene (PYR), bends (CHR), benzo (b) fluoranthene (BbF), benzo (k) fluoranthene (BkF), benzo (a) pyrene at benzo (a) anthracene (BaA)
(BaP), dibenzo (a, h) anthracene (DBA), indeno (1,2,3 ,-cd) pyrene (IPY) and benzo (g, h, i) perylene (BPE), add
Enter n-hexane, for constant volume in volumetric flask, the concentration of each ingredient is 10mgL-1.N-hexane is chromatographically pure.
According to above-mentioned chromatographic condition, using sample volume as abscissa, peak height is ordinate, draws standard curve, the mark of acquisition
Directrix curve is as shown in Figure 1, wherein and 1- naphthalene, 2- acenaphthylene, 3- acenaphthene, 4- fluorenes, 5- is luxuriant and rich with fragrance, 6- anthracene, 7- fluoranthene, 8- pyrene, 9- benzo (a) anthracene,
10- is bent, 11- benzo (b) fluoranthene, 12- benzo (k) fluoranthene, 13- benzo (a) pyrene, 14- indeno (1,2,3 ,-cd) pyrene, 15- hexichol
And (a, h) anthracene, 16- benzo (g, h, i) perylene.It is as shown in table 2 to obtain equation of linear regression.
The linear relationship table of 25 kinds of polycyclic aromatic hydrocarbons of table
Table2 The linear relationship of 5 PAHs
(5-2) detects the content of polycyclic aromatic hydrocarbon in polycyclic aromatic hydrocarbon extracting solution according to above-mentioned chromatographic condition.
(5-3) analysis detection result: testing result is shown in Table 3.
3 difference of table sprays influence of the mode to PAHs content in cabbage heart
Table3 Different spraying methods on the influence of the content of
PAHs
The blade of cabbage heart and root are respectively through 600 μ gL-1PAHs mixed solution processing after, through detecting, blade processing
When, naphthalene, phenanthrene, fluoranthene, benzo (a) anthracene and benzo (a) pyrene content be respectively 38.22 μ gkg-1、36.61μg·kg-1、
21.64μg·kg-1、19.88μg·kg-1With 10.93 μ gkg-1, the content of this 5 kinds of polycyclic aromatic hydrocarbons is respectively when Greenhouse Water Culture
3.25μg·kg-1、3.64μg·kg-1、2.67μg·kg-1、2.53μg·kg-1、1.54μg·kg-1, blade processing it is each
The content of kind polycyclic aromatic hydrocarbon is all larger than Greenhouse Water Culture group, and significant difference.In same processing mode, every kind of polycyclic aromatic hydrocarbon contains
Amount is different, all shows trend with the increase of PAHs phenyl ring number and reduced, but content when root sprays between every kind of PAHs
Difference is not significant.
Blade spraying treated ∑ PAHs content is 127.28 μ g/kg, sprays (13.63 μ g/kg) higher than root.It is right
The uptake of 5 kinds of different types of PAHs is from high to low successively are as follows: naphthalene > phenanthrene > fluoranthene > benzo (a) anthracene > benzo (a) pyrene.
Specific embodiment described herein is only an example for the spirit of the invention.The neck of technology belonging to the present invention
The technical staff in domain can make various modifications or additions to the described embodiments or replace by a similar method
In generation, however, it does not deviate from the spirit of the invention or beyond the scope of the appended claims.
Claims (3)
1. the method for inspection that a kind of polycyclic aromatic hydrocarbon enters cabbage heart main path, it is characterised in that the following steps are included:
(1) cabbage heart seedling is colonized in culture substrate, after being colonized 5 ~ 20 days, cabbage heart is at least divided into blade validation group, root
Validation group；
(2) equal weight and dense is added into the Brassica campestris leaves of blade validation group and the cabbage heart culture substrate of root validation group respectively
The polycyclic aromatic hydrocarbon mixed solution of degree continuous processing 1 ~ 6 day, grows to harvest time to cabbage heart, harvest；
(3) cabbage heart of harvest is rinsed with water completely, after vacuum refrigerating machine is dry, is crushed spare；The temperature of vacuum freeze drying
Degree is -50 ~ -40 DEG C, and pressure is 0.4 ~ 1.0MPa；
(4) smashed cabbage heart organic extraction solvent is impregnated into 15 ~ 48h, the organic extraction solvent is by methylene chloride and just
2:8 is formulated hexane by volume；Then 20 ~ 40min of ultrasonic extraction again, the temperature of ultrasonic extraction is 28 ~ 32 DEG C, using height
Fast refrigerated centrifuge centrifugation, revolving speed are 3500 ~ 4000 rpm, and temperature is 2 ~ 6 DEG C, and the time is 8 ~ 20 minutes, through Florisil silicon
Supernatant is collected after earth pillar, said extracted is repeated and operates 1 ~ 2 time, merge supernatant, be concentrated to dryness, and the temperature of concentration is 35 ~ 40
DEG C, polycyclic aromatic hydrocarbon extracting solution is obtained with 0.1~0.4 μm of aperture membrane filtration with methanol constant volume；
(5) with the content of polycyclic aromatic hydrocarbon in high performance liquid chromatograph detection polycyclic aromatic hydrocarbon extracting solution, blade validation group and root are compared
The content of polycyclic aromatic hydrocarbon in portion's validation group；
In the step (5), chromatographic condition are as follows: chromatographic column is using octadecyl silane as filler, using gradient elution, flowing
It is mutually the gradient eluent of methanol and pure water composition, column temperature is 30 DEG C, flow velocity 1ml/min；The program of the gradient elution is set as
Time gradient 0min → 3min → 5min → 30min, corresponding gradient eluent is by following volumes ratio by mobile phase A methanol and stream
Dynamic phase B pure water composition, the percentage by volume gradient 60% → 70% → 80% → 100% of mobile phase A, the volume hundred of Mobile phase B
Score gradient 40% → 30% → 20% → 0%；Ultraviolet detection wavelength: 254nm.
2. the method for inspection that polycyclic aromatic hydrocarbon according to claim 1 enters cabbage heart main path, it is characterised in that:
In the step (2), the concentration of polycyclic aromatic hydrocarbon mixed solution is 100 ~ 900 μ gL-1。
3. the method for inspection that polycyclic aromatic hydrocarbon according to claim 1 enters cabbage heart main path, it is characterised in that:
In the step (2), polycyclic aromatic hydrocarbon solution is by naphthalene, acenaphthylene, acenaphthene, fluorenes, phenanthrene, anthracene, fluoranthene, pyrene, benzo (a) anthracene, in the wrong, benzo
(b) fluoranthene, benzo (k) fluoranthene, benzo (a) pyrene, dibenzo (a, h) anthracene, indeno (1,2,3 ,-cd) pyrene and benzo (g, h, i) two
One or more of rylene ingredient is added n-hexane dissolution and is formulated.
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