CN105527235B - A kind of method of liquid-liquid extraction GFAAS methods measure monovalence copper content - Google Patents
A kind of method of liquid-liquid extraction GFAAS methods measure monovalence copper content Download PDFInfo
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Abstract
The invention belongs to trace element detection technical field, specifically discloses a kind of method of liquid-liquid extraction GFAAS methods measure monovalence copper content.The method of the present invention includes the following steps：S1. the making of standard working curve；S2. cupprous detection：Sample and isometric 20% trichloroacetic acid are mixed, according to sample copper content, the glycine NaOH buffer solutions with pH 9 are diluted by different proportion again, 0.06% (w/w) 2 of 1mL is added into mixed solution again, the n-pentanol solution of 2 ' diquinolines, mixes, stratification, take upper organic layer, upper machine graphite furnace atomic absorption spectrometry monovalence copper content.The method of the present invention can be used for the detection of biological specimen trace monovalence copper content, significant to inquiring into body copper metabolic imbalance.
Description
Technical field
The invention belongs to trace element detection technical field, and in particular, to a kind of liquid-liquid extraction-GFAAS methods measure one
The method of valency copper content.
Background technology
Copper ion is the indispensable trace element of organism.In vivo, copper mainly with enzyme, protein molecule
And the state of some biological micromolecules such as amino-acid complex exists.Some important physiology courses of copper ion participation organism,
As cellular respiration, the transmission of neurotransmitter, the intake of anti-oxidation stress and iron ion all rely on copper ion.The many diseases of human body
It is unbalance related with internal copper ion, such as hepatolenticular degeneration, rabid ox disease, alzheimer's disease and cancer.Nervus retrogression disease
Disease is related with loss of biological activity or Cu (I) toxicity of ceruloplasmin.The effect that copper ion plays in vivo is mainly base
In its redox chemical property, that is, Cu2+And Cu+Conversion between oxidation state.Some intracellular important biomolecules
Reaction is learned to be due to conversion between two oxidation state and carried out.
Organism can only quantitative determine total copper mostly at present, can not distinguish Cu (I) and Cu (II).G/L grades of biosystem μ
Trace Cu (I) ion measurement there is no document report so far both at home and abroad.In the presence of Cu (II) ion, Cu (I) copper ion is measured
It is faced with the interference problem for how eliminating Cu (II) ion.When Cu (I) ion is measured in pure chemistry system, Cu (I) ion is very
Easily aoxidized by the oxygen of air, it is very unstable in inorganic solution.But since it can form the double coordinations of high intensity with diquinoline
Base Cu (I) ionic complex, and can be stabilized in organic solvent, therefore available spectrophotometry.But to biological sample
Cu (I) ion of this trace still can not use spectrophotometry.
The content of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of side of liquid-liquid extraction-GFAAS methods measure monovalence copper content
Method.
The above-mentioned purpose of the present invention is to be achieved by the following technical programs.
A kind of method of liquid-liquid extraction-GFAAS methods measure monovalence copper content, includes the following steps：
S1. the making of standard working curve：Under the protection of inert gas, by the standard items of CuCl saturation KCl solution
Constant volume, obtains the cuprous standard solution of 1000ppm；Glycine-NaOH the buffer solutions of cuprous standard solution and pH 9 are taken by body
Product ratio 1:9 mixing, then 0.06% isometric (w/w) 2 is added into mixed solution, the n-pentanol solution of 2 '-diquinoline, is mixed
It is even, stratification, take upper organic layer to be diluted to 10 successively with the n-pentanol solution of 2,2 '-diquinoline, 1,0.1,0.01ppm it is molten
Liquid, takes machine on 0.01ppm solution 1mL, makes working curve；
S2. cupprous detection：By sample with isometric 20% trichloroacetic acid mix, according to sample copper content, then with pH
9 glycine-NaOH buffer solutions are diluted by different proportion, then 0.06% (w/w) 2,2 '-connection of 1mL is added into mixed solution
The n-pentanol solution of quinoline, mixes, stratification, takes upper organic layer, upper machine graphite furnace atomic absorption spectrometry monovalence copper
Content；
Wherein, when sample described in S2 is serum, the supernatant of sample is taken to dilute 20 times with buffer solution；Sample is described in S2
When cell liquid, cell film liquid, tissue fluid or urine, the supernatant of sample is taken to dilute 1 times with buffer solution；Sample described in S2 is not
During protein-contg inorganic solution, sample buffer solution is directly diluted 1 times.
Glycine can form the inorganic complex of stabilization with Cu (II), its stability constant is 15.1, and in pH 8-10 models
Enclose, easily generate coordination ratio 1:2 stabilization cupric ammine complex, the glycine chelate copper formed do not dissolve in organic solvent, wherein
PH is optimal when being 9.2,2 '-cuproine (2,2 '-diquinoline) is the specical regent for measuring Cu (I), with Cu (I) formation
Complex compound can be well dissolved in organic solvent and be insoluble in water.When the aqueous solution biological specimen containing glycine is with containing 2,
During the organic solvent extraction of 2 '-diquinoline, both can just be separated in water phase and organic phase well, so as to reach two
Plant ion isolation and achieve the purpose that to measure respectively.Organic phase sampling Graphite Furnace Atomic Absorption measure Cu (I) after separation, just can be big
The big sensitivity for improving measure Cu (I).
Preferably, when sample described in S2 is protein-contg sample, deproteinized is centrifuged after sample and trichloroacetic acid are mixed, then
Remove the supernatant after albumen and carry out subsequent step.Sample containing protein includes serum, cell liquid, cell film liquid, tissue fluid.
Not protein-contg inorganic solution or other biological body fluid such as urine, can reduce deproteinized step.Preferably, it is described not protein-contg
Inorganic solution includes mineral water, tap water.
Preferably, the centrifugation centrifuges 10min for 1200rpm.
Preferably, when sample described in S2 is cell liquid, cell film liquid or tissue fluid, the sample before non-deproteinized is separately taken to measure
Protein concentration, monovalence copper concentration is quantified with protein content.When sample described in S2 is cell liquid, cell film liquid or tissue fluid, because thin
Born of the same parents can not determined volume, therefore must separately take the sample before non-deproteinized to measure protein concentration, with the sample copper content of same volume divided by
Containing protein content, i.e. cuprous quality contained by every gram of albumen quantifies monovalence copper concentration.
Preferably, when sample described in S2 comes from cell, sample is pre-processed as follows：The cell after a couple of days is cultivated to receive
Collection, ultrasound or frost it is broken after, isolated supernatant, that is, cell liquid, the cell membrane proper amount of surfactant to precipitate is molten
Solution, is re-used as sample and is measured.
Preferably, the method mixed described in S1, S2 vibrates 1min for vortex.
Preferably, glycine concentration≤0.01mol/L in buffer solution described in S1, S2.
Preferably, the upper machine amount of upper organic layer described in S2 is 500 μ L.
Compared with prior art, beneficial effect of the present invention is：The present invention provides trace monovalence in a kind of biological sample
The detection method of content of copper ion, it is significant to inquiring into body copper metabolic imbalance.The method of the present invention can distinguish Cu (I) and
Cu (II), the interference of Cu (II) ion is eliminated when measuring Cu (I), the test limit of biosystem trace Cu (I) ion is reached
0.10 μ g/L, relative deviation ＜ 5%.The rate of recovery：95~102%.
Embodiment
The present invention is described in further details with reference to specific embodiment, but embodiment the present invention is not done it is any
The restriction of form.Unless stated otherwise, the reagent of the invention used, method and apparatus is the art conventional reagent, methods
And equipment.
Embodiment 1
Standard items prepare：Behind the CuCl standard items Kaifeng of 5g dresses, dispensed with 10 1mL plastic tubes, be carefully filled with argon gas, will
The mouth of pipe is sealed with fluid sealant after inner air tube drive is net, and is stored in the drier for putting silica gel.
A kind of method of liquid-liquid extraction-GFAAS methods measure monovalence copper content, includes the following steps：
S1. the making of standard working curve：When preparing working curve, wherein one pipe is taken out, weighs the standard items of CuCl
(99.999%, Aladdin) 0.0156g is placed in 10mL volumetric flasks, and graduation mark is diluted to saturation KCl solution, sub- up to 1000ppm
Cu standard solution, therefrom takes 100 μ L to be placed in 5mL plastic tubes, adds 900 μ L pH to be buffered for 9 Glycine (glycine)-NaOH molten
Liquid, then add 1000 μ L0.06%2, the n-pentanol solution of 2 '-diquinoline, vortex vibration 1min, stratification, upper strata to obtain the final product
The cuprous standard solution of 100ppm, then take the 100 μ L n-pentanol solutions of 2,2 '-diquinoline to be diluted to successively from upper organic layer
10,1,0.1,0.01ppm solution, with machine on 0.01ppm solution, n-amyl alcohol is worked curve for diluent.
S2. Cu (I) is measured：200 μ L blood serum samples and 200 μ L20% trichloroacetic acids are mixed, 1200, which leave heart 10min, goes
Albumen, takes 50 μ L of serum supernatant to put in 5mL plastic tubes, and it is molten to add the glycine Glycine-NaOH bufferings that 950 μ L pH are 9
Liquid, adds 1000 μ L0.06%2, the n-pentanol solution of 2 '-diquinoline after mixing, vortex vibrates 1min, upper strata is taken after stratification
500 μ L, upper machine graphite furnace atomic absorption spectrometry.
The sample can also be cell liquid or other biological fluids etc.；When measuring cell liquid, cell film liquid or tissue fluid,
Liquid after deproteinized presses 1 with glycine Glycine-NaOH buffer solutions：1 dilution, in addition to being measured by above-mentioned steps, separately
Certain volume sample measures protein concentration is taken, cuprous concentration is quantified with protein content；
If not protein-contg inorganic-liquid or other biological fluids such as urine, so that it may reduce deproteinized the step.When
When need to detect Cu (I) of cell membrane, cultivate the cell after a couple of days and collect, after ultrasonic or frost is broken, isolated supernatant is
Cell liquid, the cell membrane to precipitate can use proper amount of surfactant（Such as 2%Triton X-100）Dissolving, be re-used as sample into
Row measure.
According to the method described above to NongFuShanQuan, tap water, hematuria be clear, the Cu (I) in cell liquid and cell membrane is measured,
As a result such as table 1.Every group of sample repeats experiment three times.
The detection example of the different samples of table 1
Note：μg/gPro：Every gram of albumen of microgram；TCu：Total copper
This method test limit：0.10 μ g/L, relative deviation ＜ 5%.The rate of recovery：95~102%.This is to inquiring into the metabolism of human body copper
It is unbalance significant.
Supplementary notes：1, since the cuprous organic phase that has been transferred in different samples just carries out upper machine measure, thus it is all
Sample can all use same working curve to measure.
2, upper press proof liquid is generally 500 to 1000 microlitres, therefore the organic phase n-amyl alcohol containing extractant only needs plus 1mL.
Claims (8)
1. the method for a kind of liquid-liquid extraction-graphite furnace atomic absorption spectrometry monovalence copper content, it is characterised in that including following step
Suddenly：
S1. the making of standard working curve：Under the protection of inert gas, by the standard items of CuCl saturation KCl solution constant volumes,
Obtain the cuprous standard solution of 1000ppm；Take the glycine-NaOH buffer solutions of cuprous standard solution and pH9 by volume 1:9
Mixing, then 0.06% isometric (w/w) 2 is added into mixed solution, the n-pentanol solution of 2 '-diquinoline, mixes, and stands and divides
Layer, take upper organic layer to be diluted to 10 successively with the n-pentanol solution of 2,2 '-diquinoline, 1,0.1,0.01ppm solution, take
Machine on 0.01ppm solution 1mL, makes working curve；
S2. cupprous detection：Sample and isometric 20% trichloroacetic acid are mixed, according to sample copper content, then with pH9's
Glycine-NaOH buffer solutions are diluted by different proportion, then 0.06% (w/w) 2,2 '-connection quinoline of 1mL is added into mixed solution
The n-pentanol solution of quinoline, mixes, and stratification takes upper organic layer, and upper machine graphite furnace atomic absorption spectrometry monovalence copper contains
Amount；
Wherein, when sample described in S2 is serum, the supernatant of sample is taken to dilute 20 times with buffer solution；Sample described in S2 is cell
When liquid, cell film liquid, tissue fluid or urine, the supernatant of sample is taken to dilute 1 times with buffer solution；Sample described in S2 is without egg
During white inorganic solution, sample buffer solution is directly diluted 1 times.
2. according to the method described in claim 1, it is characterized in that, when sample described in S2 is protein-contg sample, by sample with
Trichloroacetic acid centrifuges deproteinized after mixing, then removes the supernatant after albumen and carry out subsequent step.
3. according to the method described in claim 2, it is characterized in that, the centrifugation centrifuges 10min for 1200rpm.
4. according to the method described in claim 2, it is characterized in that, sample described in S2 is cell liquid, cell film liquid or tissue fluid
When, separately take the sample before non-deproteinized to measure protein concentration, monovalence copper concentration is quantified with protein content.
5. according to the method described in claim 2, it is characterized in that, when sample described in S2 comes from cell, sample is carried out as follows
Pretreatment：Cultivate the cell after a couple of days to collect, after ultrasonic or frost is broken, isolated supernatant, that is, cell liquid, precipitates
Cell membrane dissolved with proper amount of surfactant, be re-used as sample and be measured.
6. according to the method described in claim 1, it is characterized in that, the method mixed described in S1, S2 vibrates 1min for vortex.
7. according to the method described in claim 1, it is characterised in that glycine concentration in buffer solution described in S1, S2≤
0.01mol/L。
8. according to the method described in claim 1, it is characterized in that, the upper machine amount of upper organic layer described in S2 is 500 μ L.
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| CN101169424A (en) * | 2006-10-24 | 2008-04-30 | 苏州艾杰生物科技有限公司 | Copper diagnosis/determination reagent kit and copper concentration determination method |
| CN101865854A (en) * | 2010-06-04 | 2010-10-20 | 广东环凯微生物科技有限公司 | Reagent for quickly determining copper concentration in water and use method thereof |
| CN102095724A (en) * | 2010-11-24 | 2011-06-15 | 青岛佳明测控仪器有限公司 | Method for determining copper content in water by biquinolyl spectrophotometry |
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| PL342328A1 (en) * | 2000-09-01 | 2002-03-11 | Kghm Polska Miedz Sa | Method fo measuring concentration of copper ions in industrial electrolytes |
| US20080090297A1 (en) * | 2006-10-13 | 2008-04-17 | Novus International Inc. | Methods for determining the metal bioavailability of metal sources |
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| CN101169424A (en) * | 2006-10-24 | 2008-04-30 | 苏州艾杰生物科技有限公司 | Copper diagnosis/determination reagent kit and copper concentration determination method |
| CN101865854A (en) * | 2010-06-04 | 2010-10-20 | 广东环凯微生物科技有限公司 | Reagent for quickly determining copper concentration in water and use method thereof |
| CN102095724A (en) * | 2010-11-24 | 2011-06-15 | 青岛佳明测控仪器有限公司 | Method for determining copper content in water by biquinolyl spectrophotometry |
Non-Patent Citations (3)
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| GFAAS测定乙醇-EDTA法分离血清中不同形态的铜和锌;张源 等;《光谱学与光谱分析》;20100331;第30卷(第3期);816-819 * |
| Spectrofotometric determination of copper in sugar cane spirit using biquinoline in the presence of ethanol and Triton X-100;Sarah Adriana do Nascimento Rocha 等;《Spectrochimica Acta Part A》;20081231;第71卷;1414-1418 * |
| 超声透析-GFAAS法测定动物组织可溶性小分子蛋白结合铜;张源 等;《分析化学》;20131130;第41卷(第11期);1784-1785 * |
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