CN103512872A - Colorimetric analysis method for quantitatively detecting copper ions through fluorescent equipment - Google Patents
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Abstract
The invention relates to fluorescent colorimetric analysis and discloses a colorimetric analysis method for quantitatively detecting copper ions through fluorescent equipment. The colorimetric analysis method comprises the following steps: reacting a copper ion solution and a Rhodamine fluorescent indicator synthesized in a lab to enhance fluorescence, and exciting the indicator through light with a particular wavelength of 365nm by adopting home-made fluorescent colorimetric equipment; catching fluorescence color change of the indicator before and after the copper ions are added by using electronic imaging equipment such as a scanner and a digital camera; extracting color change values of red, green and blue (RGB) channels in the image before and after color change, and establishing a standard curve between the copper ions and a concentration gradient which corresponds to the copper ions; and comparing the obtained color change values of the sample with values in the curve in a sample test process, thus quantitatively and selectively analyzing the copper ions in an unknown sample. According to the method, copper ion concentration of 10<-5>M can be detected, co-existing interference of nine ions can be effectively eliminated, and the method has high selectivity.
Description
Technical field
The present invention relates to fluorescence colorimetric method, a kind of colorimetric analysis method that specifically fluorescence equipment quantitatively detects for copper ion.
Background technology
Copper, because of its good physical strength, good ductility, superpower memory power, and stable chemical property and be widely used in iron and steel, metallurgy, paint, national defence, electronics, chemistry and the industry such as ceramic, brought revolutionary change to production.The pollution of various copper is also lost in environment thereupon, brings threat to people's life.Though the copper necessary metallic element that is human life activity, possible copper requirement is 25-35 μ g for each person every day, and copper participates in composition and the metabolism of enzyme in vivo, can promote insulin secretion, reduces blood sugar.Yet the harm that excessive copper causes human body can not be ignored really.The absorption of excess copper can cause the property of cardiac muscle, brain, liver lung and renal function to become, even cause cancer.Therefore, in each field of technical analysis, environment measuring, drug screening and life science, all need the content of copper to measure.China has carried out clearly regulation to the content of daily copper in drinking water ion in new " drinking water sanitary standard ", can not exceed 1mg/L.
The copper ion qualitative and quantitative detection technology extensively adopting at present in the world comprises: spectrophotometric method, atomic absorption spectrography (AAS), inductively coupled plasma emission spectrography, inductively coupled plasma mass spectrometry, anodic stripping voltammetry, plasma induction light spectrometry etc.
These methods can successfully detect copper ion pollutant in water, but general cost is higher, need large-scale instrument and skilled operating personnel, and generally sampling at the scene, then deliver to laboratory and carry out off-line analysis, exist consuming timely, analytical procedure is complicated, analytical instrument is expensive, and the low and sample of sample frequency is difficult for the shortcomings such as preservation.In some cases, need to know in time environmental pollution situation, to formulate rapidly corresponding treatment Countermeasures.In the face of these difficult problems, optimal solution is exactly that original position detects in real time.Also have on the market at present some commercial copper ion test papers, some is taked to infiltrate to contain the organic solution of indicator and dry and makes, and does not take into full account the fixing of indicator, causes test paper easily to decolour; Other adopt in manufacturing test paper process indicator are mixed and is prepared with paper pulp, and manufacturing process is loaded down with trivial details, and detection sensitivity is not satisfactory.
Summary of the invention
A kind of colorimetric analysis method that the object of the present invention is to provide fluorescence equipment quantitatively to detect for copper ion.
For achieving the above object, the technical solution used in the present invention is:
The colorimetric analysis method quantitatively detecting for copper ion, comprises the synthetic of rhodamine quinolines fluorescence indicator, the chelating of copper ion, and the building of fluorescence colorimetric equipment, fluorescence colorimetric reads, and sample determination, it is characterized in that:
The sequestering power to metallic ion in conjunction with oxine and rhodamine indicator, the bright quinolines fluorescence indicator of synthesizing rhodamine, detects the copper ion of variable concentrations; Adopt fluorescence colorimetric equipment, change in fluorescence image by digital camera after to copper ion and indicator reaction gathers, the change color value of extracting three passages of red, green, blue (RGB) in its change color front and back image, builds the typical curve that copper ion is answered concentration gradient in contrast;
1) rhodamine quinolines indicator is synthetic: adopt simple one-step synthesis, by rhodamine B hydrazides 2. with oxine-2-formaldehyde reaction, generate rhodamine quinolines copper ion fluorescence indicator 1.:
2) chelating of copper ion: the solution (5 * 10 of the 1mL copper ion fluorescence indicator obtaining to step 1)
-4m) in, add respectively 1.6 * 10 of 12.5 μ L
-3m, 3.2 * 10
-3m, 4.8 * 10
-3m, 6.4 * 10
-3m, 8 * 10
-3m, 1.6 * 10
-2m, 4 * 10
-2m, 8 * 10
-2the copper ion solution of M, puts it into isothermal reaction 30min(± 2min in the water-bath of 70 ℃ (± 2 ℃)) after, the color of solution becomes redness by lightpink, and solution tends towards stability;
3) building of fluorescence colorimetric equipment: select 360-410nm light emitting diode, obtain more single 365nm light source as fluorescence burst of ultraviolel light source after filtering via the narrow band pass filter that can allow 365nm light pass through; Get 200 microlitre steps 2) solution that obtains after reaction is placed in transparent plastic tubule, adopt this excitation source to rhodamine quinolines indicator with and excite with the reacted solution of variable concentrations copper ion;
4) fluorescence colorimetric reads: the solution fluorescence color after via digital imaging device, step 3) being excited carries out imaging from tubule bottom, color, by after the digitizing of Photoshop software, is extracted to red (R), green (G) in the image of change color front and back, the change color value (⊿ R=R of blue (B) three passages
after reaction-R
before reaction， ⊿ G=G
after reaction-G
before reaction， ⊿ B=B
after reaction-B
before reaction), build corresponding concentration-gradient curve with copper ion concentration;
5) sample determination: by the solution (5 * 10 of the rhodamine quinolines copper ion fluorescence indicator of the copper ion sample of 12.5 μ L unknown concentration and step 1) acquisition
-4m) after 1mL reaction, carry out imaging analysis, the concentration-gradient curve of its upper foundation of the change color value substitution obtaining, tries to achieve copper ion concentration.
Method according to claim 1, is characterized in that: described copper ion indicator is rhodamine quinolines fluorescence indicator, combines rhodamine and the oxine chelation to metallic ion.
Method according to claim 1, is characterized in that: described digital imaging device is color-image forming apparatus, as CCD, digital camera or scanner.
Method according to claim 1, is characterized in that: indicator adds the reacted color of copper ion can be reflected by digitized form via Photoshop software, for quantitative test provides foundation.
Method according to claim 1, is characterized in that: described plastics tubule is originally cylindrical as water white transparency, its internal diameter 6mm, and external diameter 8mm, high 12mm, its pipe end light transmission is good.Method according to claim 1, it is characterized in that: the light emitting diode uviol lamp series connection that described fluorescence excitation equipment is 8 4V 20mA, and it is fixed on to the black cover bucket central authorities of a top seal uniformly, cover bucket lower ending opening, form an area source, light emitting diode below is fixed with the narrow band pass filter that only can allow 365nm light pass through that a diameter is 2.5cm, makes the light seeing through only have 365nm left and right; In addition, putting the bandpass filter of being furnished with another 400-700nm below the tubule bottom of reaction solution, be used for filtering out the bias light that LED light lamp is sent.
According to the method described in claim 1 or 4, it is characterized in that: after sample test process, extraction indicator adds the image before and after copper ion, adopt Photoshop software by after image digitazation intuitively, extract red (R), green (G) in the image of change color front and back, the change color value (⊿ R=R of blue (B) three passages
after reaction-R
before reaction， ⊿ G=G
after reaction-G
before reaction， ⊿ B=B
after reaction-B
before reaction), by the concentration-gradient curve of change color value and concentration gradient structure copper ion.
Preparation feedback solution, specifically preparation according to the following procedure:
1) preparation of indicator solution: synthetic rhodamine quinolines fluorescence indicator is dissolved in ethanolic solution according to certain massfraction, is then used second alcohol and water dilution (volume ratio: alcohol/water=7/3), the final concentration that makes indicator is 5 * 10
-5~ 5 * 10
-4m;
2) preparation of copper standard solution: adopt Wahaha drinking pure water, compound concentration 1.6 * 10
-3m, 3.2 * 10
-3m, 4.8 * 10
-3m, 6.4 * 10
-3m, 8 * 10
-3m, 1.6 * 10
-2m, 4 * 10
-2m, 8 * 10
-2a series of copper ion standard solution of M;
3) reacting of indicator and copper ion: the above-mentioned indicator solution in 1. of 0.1-1.5mL is joined in 1.5mL plastics tubule, the copper standard solution 2-25 μ L getting 2. joins in plastics tubule, fully mix to be placed in 30-70 ℃ of thermostat water bath and react 5-30 minute, obtain the red solution that reaction tends towards stability;
4) design of fluorescence excitation equipment: fluorescence excitation equipment comprises the light emitting diode uviol lamp series connection of 8 4V 20mA, and it is fixed on to the black cover bucket central authorities of a top seal uniformly, cover bucket lower ending opening, form an area source, light emitting diode below is fixed with the narrow band pass filter that only can allow 365nm light pass through that a diameter is 2.5cm, makes the light seeing through only have 365nm left and right; In addition, putting the bandpass filter of being furnished with another 400-700nm below the tubule bottom of reaction solution, be used for filtering out the bias light that LED light lamp is sent.
5) imaging colorimetric: in the color-image forming apparatus extraction plastics tubules such as employing CCD, digital camera or scanner, indicator adds the color of copper ion front and back, passes through Photoshop
tMdeng software, the color producing before and after membrane filtration is carried out to digitized processing, obtain rgb value corresponding to indicator reaction front and back image, by not adding the rgb value of image after concentration copper ion and indicator reaction, deduct the rgb value that does not add image before copper ion, obtain the Δ R of " the poor image that subtracts ", Δ G, with Δ B value, by three's quadratic sum again the value of evolution build normal concentration curve with corresponding copper ion concentration of standard solution, for the analysis to unknown concentration copper solution.
Tool of the present invention has the following advantages:
1. the method is reacted with fluorescence indicator and can be made fluorescence strengthen this phenomenon according to copper ion, and the indicator color before and after reacting with copper ion is contrasted, and gathers image, by its digitizing by colored digital equipment.By after color digitizing intuitively, make quantitative measurement become possibility, also further promoted sensitivity simultaneously, the change color that small naked eyes are difficult to see can display more intuitively by the difference after digitizing.
2. fluorescence excitation used and image capture device are simple, and light being easy to carry about with one can be used for on-the-spot test sample, and image acquisition is rapid, can detect in real time.
3. the foundation of normal concentration curve, making becomes possibility to the accurate quantification of unknown sample concentration; Got rid of single dependence colorimetric card, naked eyes are estimated the defect of approximate concentration.
Accompanying drawing explanation
Fig. 1 is the structural representation of fluorescence excitation equipment; Select 360-410nm light emitting diode (101), after filtering via 365nm narrow band pass filter (102), obtain more single 365nm light source as fluorescence burst of ultraviolel light source; Get 200 microlitre reaction solutions and be placed in transparent plastic tubule (201), tubule is placed in to mould deck (301), in order to the fixing position of tubule, be beneficial to imaging comparison.Adopt the above 365nm excitation source obtaining to rhodamine quinolines indicator with and the light beam that excites ，Zhong interval to send in order to homogenization light emitting diode with quartz wool glass (501) with the reacted solution of variable concentrations copper ion.Tubule below is embedded in the 400-700nm bandpass filter (401) in black sleeve (402) with another, is used for the bias light of filtering light emitting diode, and digital camera is from the bottom imaging of black sleeve.
Fig. 2 is standard color comparison card and normal concentration curve.Comprising three independent concentration curves of passage of red, green, blue, and square add and the total change color curve after evolution again.
Fig. 3 is various coexisting ions (9 metal ion species: Pb
2+, Hg
2+, Cd
2+, Zn
2+, Ca
2+, Co
2+, Fe
3+, Hg
2+, Ni
2+) under disturbed condition, copper ion solution (5 * 10
-4m) red, the turquoise passage Δ R after the colorimetric estimation of the method fluorescence, Δ G, total change color value of Δ B.With respect to there is no coexisting ion disturbed condition, be less than 6% relative deviation and confirmed the selectivity that the method is fabulous.
Embodiment
The foundation of standard color comparison card and normal concentration curve
Be specially:
1) preparation of indicator solution: rhodamine fluorescence indicator is dissolved in ethanolic solution according to certain massfraction, is then used second alcohol and water dilution (volume ratio: alcohol/water=7/3), the final concentration that makes indicator is 5 * 10
-4m.
2) preparation of copper standard solution: adopt Wahaha drinking pure water, respectively compound concentration 1.6 * 10
-3m, 3.2 * 10
-3m, 4.8 * 10
-3m, 6.4 * 10
-3m, 8 * 10
-3m, 1.6 * 10
-2m, 4 * 10
-2m, 8 * 10
-2a series of copper ion standard solution of M;
3) reacting of indicator and copper ion: the above-mentioned indicator solution in 1. of 1mL is joined in 1.5mL plastics tubule, the copper standard solution 12.5 μ L that get 2. join in plastics tubule, fully mix to be placed in 70 ° of C thermostat water baths and react 30 minutes, obtain the red solution that reaction tends towards stability.
4) design of fluorescence excitation equipment: fluorescence excitation equipment comprises the light emitting diode uviol lamp series connection of 8 4V 20mA, and it is fixed on to the black cover bucket central authorities of a top seal uniformly, cover bucket lower ending opening, form an area source, light emitting diode below is fixed with the narrow band pass filter that only can allow 365nm light pass through that a diameter is 2.5cm, makes the light seeing through only have 365nm left and right; In addition, putting the bandpass filter of being furnished with another 400-700nm below the tubule bottom of reaction solution, be used for filtering out the bias light that LED light lamp is sent;
5) imaging colorimetric: in the color-image forming apparatus extraction plastics tubules such as employing CCD, digital camera or scanner, indicator adds the color of copper ion front and back, passes through Photoshop
tMdeng software, the color producing before and after membrane filtration is carried out to digitized processing, obtain rgb value corresponding to indicator reaction front and back image, by not adding the rgb value of image after concentration copper ion and indicator reaction, deduct the rgb value that does not add image before copper ion, obtain the Δ R of " the poor image that subtracts ", Δ G, with Δ B value, by three's quadratic sum again the value of evolution build normal concentration curve (as shown in Figure 2) with corresponding copper ion concentration of standard solution.
The mensuration of unknown sample:
Preparation unknown concentration copper ion sample solution, uses with the syringe of 0.45 ± 0.2 μ m micropore nylon leaching film filtering head and draws 50-100mL testing sample, to remove organism in sample, and bulky grain or cotton-shaped impurity; Remove filtering head, solution is transferred in beaker; With 0.5M watery hydrochloric acid or sodium hydroxide solution, regulating the pH value of the rear sample solution of filtration is 6-8.
Get the above-mentioned solution of 12.5 microlitre, join 1mL containing 5 * 10
-4the solution of M copper ion indicator, shakes up to be placed in 70 ℃ of thermostat water baths and reacts 30min;
Adopt above-mentioned same fluorescence to get the equipment of elephant, the solution after stable is got and resembled; Its color and standard color comparison card are compared, drawn roughly concentration.Red, green, blue (RGB) value of extracting its change color front and back differs from and subtracts, and obtains the Δ R of modified-image, Δ G, Δ B value.By Δ R, Δ G, the value of Δ B and three's quadratic sum be again in the value of evolution (13.59) substitution copper standard solution curve, by typical curve linear equation (shown in Fig. 2, y=157.62x-0.52), try to achieve the copper solution concentration (8.95 * 10 accurate to be measured of answering in contrast
-5m).
The present invention adopts homemade fluorescence colorimetric equipment, and the ultraviolet light by 365nm specific wavelength excites indicator.With electronic imaging device, as adding the fluorescence color of front and back indicator, the seizure copper ions such as scanner, digital camera change.The change color value of extracting three passages of red, green, blue (RGB) in its change color front and back image, builds the typical curve that copper ion is answered concentration gradient in contrast.By after color digitizing intuitively, make quantitative measurement become possibility, also further promoted sensitivity simultaneously, the change color that small naked eyes are difficult to see can display more intuitively by the difference after digitizing.Fluorescence excitation and image capture device used are simple, and light being easy to carry about with one can be used for on-the-spot test sample, and image acquisition is rapid, can detect in real time.The method can detect 10
-5the ion concentration of M, and can effectively get rid of 9 kinds of ion coexists and disturb, higher selectivity there is.
Claims (7)
1. the colorimetric analysis method that fluorescence equipment quantitatively detects for copper ion, comprises the synthetic of rhodamine quinolines fluorescence indicator, the chelating of copper ion, and the building of fluorescence colorimetric equipment, fluorescence colorimetric reads, and sample determination, it is characterized in that:
The sequestering power to metallic ion in conjunction with oxine and rhodamine indicator, the bright quinolines fluorescence indicator of synthesizing rhodamine, detects the copper ion of variable concentrations; Adopt fluorescence colorimetric equipment, change in fluorescence image by digital camera after to copper ion and indicator reaction gathers, the change color value of extracting three passages of red, green, blue (RGB) in its change color front and back image, builds the typical curve that copper ion is answered concentration gradient in contrast;
1) rhodamine quinolines indicator is synthetic: adopt simple one-step synthesis, by rhodamine B hydrazides 2. with oxine-2-formaldehyde reaction, generate rhodamine quinolines copper ion fluorescence indicator 1.:
2) chelating of copper ion: the solution (5 * 10 of the 1mL copper ion fluorescence indicator obtaining to step 1)
-4m) in, add respectively 1.6 * 10 of 12.5 μ L
-3m, 3.2 * 10
-3m, 4.8 * 10
-3m, 6.4 * 10
-3m, 8 * 10
-3m, 1.6 * 10
-2m, 4 * 10
-2m, 8 * 10
-2the copper ion solution of M, puts it into isothermal reaction 30min(± 2min in the water-bath of 70 ℃ (± 2 ℃)) after, the color of solution becomes redness by lightpink, and solution tends towards stability;
3) building of fluorescence colorimetric equipment: select 360-410nm light emitting diode (light emitting diode), obtain more single 365nm light source as fluorescence burst of ultraviolel light source after filtering via the narrow band pass filter that can allow 365nm light pass through; Get 200 microlitre steps 2) solution that obtains after reaction is placed in transparent plastic tubule, adopt this excitation source to rhodamine quinolines indicator with and excite with the reacted solution of variable concentrations copper ion;
4) fluorescence colorimetric reads: the solution fluorescence color after via digital imaging device, step 3) being excited carries out imaging from tubule bottom, color, by after the digitizing of Photoshop software, is extracted to red (R), green (G) in the image of change color front and back, change color value (the Δ R=R of blue (B) three passages
after reaction-R
before reaction, Δ G=G
after reaction-G
before reaction, Δ B=B
after reaction-B
before reaction), build corresponding concentration-gradient curve with copper ion concentration;
5) sample determination: by the solution (5 * 10 of the rhodamine quinolines copper ion fluorescence indicator of the copper ion sample of 12.5 μ L unknown concentration and step 1) acquisition
-4m) after 1mL reaction, carry out imaging analysis, the concentration-gradient curve of its upper foundation of the change color value substitution obtaining, tries to achieve copper ion concentration.
2. method according to claim 1, is characterized in that: described copper ion indicator is rhodamine quinolines fluorescence indicator, combines rhodamine and the oxine chelation to metallic ion.
3. method according to claim 1, is characterized in that: described digital imaging device is color-image forming apparatus, as CCD, digital camera or scanner.
4. method according to claim 1, is characterized in that: indicator adds the reacted color of copper ion can be reflected by digitized form via Photoshop software, for quantitative test provides foundation.
5. method according to claim 1, is characterized in that: described plastics tubule is originally cylindrical as water white transparency, its internal diameter 6mm, and external diameter 8mm, high 12mm, its pipe end light transmission is good.
6. method according to claim 1, it is characterized in that: the light emitting diode uviol lamp series connection that described fluorescence excitation equipment is 8 4V20mA, and it is fixed on to the black cover bucket central authorities of a top seal uniformly, cover bucket lower ending opening, form an area source, light emitting diode below is fixed with the narrow band pass filter that only can allow 365nm light pass through that a diameter is 2.5cm, makes the light seeing through only have 365nm left and right; In addition, putting the bandpass filter of being furnished with another 400-700nm below the tubule bottom of reaction solution, be used for filtering out the bias light that LED light lamp is sent.
7. according to the method described in claim 1 or 4, it is characterized in that: after sample test process, extraction indicator adds the image before and after copper ion, adopt Photoshop software by after image digitazation intuitively, extract red (R), green (G) in the image of change color front and back, change color value (the Δ R=R of blue (B) three passages
after reaction-R
before reaction, Δ G=G
after reaction-G
before reaction, Δ B=B
after reaction-B
before reaction), by the concentration-gradient curve of change color value and concentration gradient structure copper ion.
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