CN103512872B - A kind of fluorescence equipment is for the colorimetric methods of copper ion detection by quantitative - Google Patents
A kind of fluorescence equipment is for the colorimetric methods of copper ion detection by quantitative Download PDFInfo
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Abstract
The present invention relates to fluorometric analysis, it is a kind of fluorescence equipment colorimetric methods for copper ion detection by quantitative: the rhodamine fluorescence indicator that copper ion solution and laboratory synthesize reacts and can make its Fluorescence Increasing, adopt homemade fluorescence colorimetric equipment, by the light of 365nm specific wavelength, indicator is excited.With electronic imaging device, add the fluorescence color change of front and back indicator as scanner, digital camera etc. catch copper ion.Extract the color changing value of red, green, blue (RGB) three passages in the image of its color change front and back, build copper ion and correspond the standard curve of Concentraton gradient.In sample test process, by the color changing value of gained sample and the value comparison in curve, thus the copper ion in unknown sample being carried out quantitative selectivity analysis.The method can detect 10-5The copper ion concentration of M, and can effectively get rid of 9 kinds of ion mutual interferences, there is higher selectivity.
Description
Technical field
The present invention relates to fluorescence colorimetric method, specifically a kind of fluorescence equipment is for the colorimetric methods of copper ion detection by quantitative.
Background technology
Copper, is widely used in iron and steel, metallurgy, paint, national defence, electronics, the industry such as chemical and ceramic, brings revolutionary change to production because of the mechanical strength that it is excellent, good ductility, superpower memory power, and stable chemical property.The pollution of various copper is also lost in environment therewith, brings threat to the life of people.Though copper is metallic element necessary to human life activity, copper requirement possible for each person every day is 25-35 μ g, and copper participates in composition and the metabolism of enzyme in vivo, can promote insulin secretion, reduces blood glucose.But, the harm that human body is caused by excessive copper can not be ignored really.The absorption of excess copper may result in the property of cardiac muscle, brain, liver lung and renal function and becomes, even cause cancer.Therefore, all need the content of copper is measured in each field of Industrial Analysis, environment measuring, drug screening and life sciences.China in new " drinking water sanitary standard " to daily drunk water in the content of copper ion carried out clear stipulaties, 1mg/L can not be exceeded.
Copper ion qualitative and quantitative detection technology widely used at present in the world includes: spectrophotography, atomic absorption spectrography (AAS), inductively coupled plasma emission spectrography, inductively coupled plasma mass spectrometry, anodic stripping voltammetry, plasma sensing spectrographic method etc..
These methods can successfully detect copper ion pollutant in water, but it is generally relatively costly, need large-scale instrument and skilled operator, and it is typically in spot sampling, being then delivered to laboratory and carry out off-line analysis, exist consuming time, analytical procedure is complicated, analytical tool is expensive, and sample frequency is low and the shortcoming such as sample not easily preservation.In some cases, it is necessary to know environmental pollution situation in time, in order 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 having some commercial copper ion test papers on the market at present, some is taked the infiltration organic solution containing indicator and dries prepared, does not take into full account the fixing of indicator, causes that reagent paper easily decolours；Other adopt indicator to be mixed with paper pulp in manufacturing reagent paper process and are prepared, and manufacturing process is loaded down with trivial details, and detection sensitivity is not satisfactory.
Summary of the invention
It is an object of the invention to provide a kind of fluorescence equipment colorimetric methods for copper ion detection by quantitative.
For achieving the above object, the technical solution used in the present invention is:
A kind of fluorescence equipment is for the colorimetric methods of copper ion detection by quantitative, and including the synthesis of rhodamine quinolines fluorescence indicator, the chelating of copper ion, building of fluorescence colorimetric equipment, fluorescence colorimetric reads, and sample determination, it is characterised in that:
In conjunction with oxine and the rhodamine indicator sequestering power to metal ion, the bright quinolines fluorescence indicator of synthesizing rhodamine, the copper ion of variable concentrations is detected；Adopt fluorescence colorimetric equipment, by digital camera, the change in fluorescence image after copper ion and indicator reaction is acquired, extract the color changing value of red, green, blue (RGB) three passages in the image of its color change front and back, build copper ion and correspond the standard curve of Concentraton gradient；
1) synthesis of rhodamine quinolines indicator: 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: to the solution (5 × 10 of the 1mL copper ion fluorescence indicator that step 1) obtains-4M) the 1.6 × 10 of 12.5 μ L it are separately added in-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 DEG C (± 2 DEG C)) after, the color from light complexion changed of solution becomes redness, and solution tends towards stability；
3) the building of fluorescence colorimetric equipment: select 360-410nm light emitting diode, obtains more single 365nm light source as fluorescent ultraviolet excitation source after filtering via the narrow band pass filter that 365nm light can be allowed to pass through；Taking 200 microlitre steps 2) solution that obtains after reaction is placed in transparent plastic tubule, adopts this excitation source that rhodamine quinolines indicator and itself and the reacted solution of variable concentrations copper ion are excited；
4) fluorescence colorimetric reads: the solution fluorescence color after step 3) being excited via digital imaging device carries out imaging bottom tubule, by color by, after Photoshop software digital, extracting the color changing value (R=R of red (R), green (G) in the image of color change front and back, blue (B) three passagesAfter reaction-RBefore reaction, G=GAfter reaction-GBefore reaction, B=BAfter reaction-BBefore 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 that the copper ion sample of 12.5 μ L unknown concentration obtains with step 1)-4M) after 1mL reaction, carrying out imaging analysis, the color changing value obtained substitutes into the concentration-gradient curve set up on it, tries to achieve copper ion concentration.
Method according to claim 1, it is characterised in that: described copper ion indicator is rhodamine quinolines fluorescence indicator, combines rhodamine and the oxine chelation to metal ion.
Method according to claim 1, it is characterised in that: described digital imaging device is color-image forming apparatus, such as CCD, digital camera or scanner.
Method according to claim 1, it is characterised in that: indicator is added the reacted color of copper ion and via Photoshop software, can be reflected by digitized form, provides foundation for quantitative analysis.
Method according to claim 1, it is characterised in that: described plastic syringe is originally cylindrical as water white transparency, and its internal diameter 6mm, external diameter 8mm, high 12mm, light transmission at the bottom of its pipe is good.Method according to claim 1, it is characterized in that: described fluorescence excitation equipment is the light emitting diode uviol lamp series connection of 8 4V20mA, and the black that it is fixed on a top seal uniformly overlaps bucket central authorities, set bucket lower ending opening, constitute an area source, it is fixed with the narrow band pass filter that 365nm light only can be allowed to pass through that diameter is 2.5cm below light emitting diode, makes the light passed through only have about 365nm；It addition, be furnished with the bandpass filter of another 400-700nm at the tubule bottom part down putting reaction solution, it is used for filtering out the bias light that LED light lamp sends.
Method according to 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 after image digitazation intuitively, the color changing value (R=R of red (R), green (G) in the image of color change front and back, blue (B) three passages will to be extractedAfter reaction-RBefore reaction, G=GAfter reaction-GBefore reaction, B=BAfter reaction-BBefore reaction), the concentration-gradient curve of copper ion is built by color changing value and Concentraton gradient.
Prepare reaction solution, specifically prepare according to the following procedure:
1) preparation of indicator solution: be dissolved in alcoholic solution according to certain mass fraction by the rhodamine quinolines fluorescence indicator of synthesis, is then used second alcohol and water dilution (volume ratio: alcohol/water=7/3), is made final concentration of the 5 × 10 of indicator-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) reaction of indicator and copper ion: by above-mentioned for 0.1-1.5mL 1. in indicator solution join in 1.5mL plastic syringe, take copper standard solution 2-25 μ L 2. and join in plastic syringe, fully mixing is placed in 30-70 DEG C of thermostat water bath and reacts 5-30 minute, obtain reacting the red solution tended towards stability；
4) design of fluorescence excitation equipment: fluorescence excitation equipment includes the light emitting diode uviol lamp series connection of 8 4V20mA, and the black that it is fixed on a top seal uniformly overlaps bucket central authorities, set bucket lower ending opening, constitute an area source, it is fixed with the narrow band pass filter that 365nm light only can be allowed to pass through that diameter is 2.5cm below light emitting diode, makes the light passed through only have about 365nm；It addition, be furnished with the bandpass filter of another 400-700nm at the tubule bottom part down putting reaction solution, it is used for filtering out the bias light that LED light lamp sends.
5) imaging colorimetric: adopt the color-image forming apparatus such as CCD, digital camera or scanner to extract indicator in plastic syringe and add the color before and after copper ion, pass through PhotoshopTMIt is digitized processing to the color produced before and after membrane filtration Deng software, obtain the rgb value that image before and after indicator reaction is corresponding, the rgb value of image after being not added with concentration copper ion and indicator reaction is deducted and does not add the rgb value of image before copper ion, obtain the Δ R of " subtractive image ", Δ G, with Δ B value, the value of three's quadratic sum evolution again and corresponding copper ion concentration of standard solution are built standard concentration curve, for the analysis to unknown concentration copper solution.
Present invention have the advantage that
1. the method is reacted according to copper ion and fluorescence indicator and can be made this phenomenon of Fluorescence Increasing, the indicator color before and after reacting with copper ion is contrasted, gathers image by colored digital equipment, by its digitized.After color digitized intuitively so that quantitative assay is possibly realized, also improving sensitivity further, the color change that small naked eyes are difficult to see can be displayed more intuitively by the difference after digitized simultaneously.
2. fluorescence excitation used by and image capture device are simple, light are easy to carry about with one, and can be used for on-the-spot test sample, and image acquisition is rapid, it is possible to detect in real time.
3. the foundation of standard concentration curve so that the accurate quantification of unknown sample concentration is possibly realized；Eliminating single dependence colorimetric card, naked eyes estimate 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 fluorescent ultraviolet excitation source；Take 200 microlitre reaction solutions to be placed in transparent plastic tubule (201), tubule is placed in mould deck (301), in order to the position of fixing tubule, is beneficial to imaging comparison.Adopt 365nm excitation source derived above that rhodamine quinolines indicator and itself and the reacted solution of variable concentrations copper ion are excited, the light beam that middle interval sends in order to homogenization light emitting diode with quartz wool glass (501).Being embedded in the 400-700nm bandpass filter (401) in black sleeve (402) with another below tubule, be used for filtering the bias light of light emitting diode, digital camera is from black sleeve bottom imaging.
Fig. 2 is standard color comparison card and standard concentration curve.Including the concentration curve that three passages of red, green, blue are independent, and square add and total color change curve after evolution again.
Fig. 3 is various coexisting ion (9 metal ion species: Pb2+,Hg2+,Cd2+,Zn2+,Ca2+,Co2+,Fe3+,Hg2+,Ni2+) under disturbed condition, copper ion solution (5 × 10-4M) total color changing value of red, turquoise passage Δ R, Δ G, the Δ B after the method fluorescence colorimetric determination.Relative to not having coexisting ion disturbed condition, the relative deviation less than 6% confirms the selectivity that the method is fabulous.
Detailed description of the invention
The foundation of standard color comparison card and standard concentration curve
Particularly as follows:
1) preparation of indicator solution: be dissolved in alcoholic solution according to certain mass fraction by rhodamine fluorescence indicator, is then used second alcohol and water dilution (volume ratio: alcohol/water=7/3), is made final concentration of the 5 × 10 of indicator-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) reaction of indicator and copper ion: by above-mentioned for 1mL 1. in indicator solution join in 1.5mL plastic syringe, take copper standard solution 12.5 μ L 2. and join in plastic syringe, fully mixing is placed in 70 ° of C thermostat water baths and reacts 30 minutes, obtain reacting the red solution tended towards stability.
4) design of fluorescence excitation equipment: fluorescence excitation equipment includes the light emitting diode uviol lamp series connection of 8 4V20mA, and the black that it is fixed on a top seal uniformly overlaps bucket central authorities, set bucket lower ending opening, constitute an area source, it is fixed with the narrow band pass filter that 365nm light only can be allowed to pass through that diameter is 2.5cm below light emitting diode, makes the light passed through only have about 365nm；It addition, be furnished with the bandpass filter of another 400-700nm at the tubule bottom part down putting reaction solution, it is used for filtering out the bias light that LED light lamp sends；
5) imaging colorimetric: adopt the color-image forming apparatus such as CCD, digital camera or scanner to extract indicator in plastic syringe and add the color before and after copper ion, pass through PhotoshopTMIt is digitized processing to the color produced before and after membrane filtration Deng software, obtain the rgb value that image before and after indicator reaction is corresponding, the rgb value of image after being not added with concentration copper ion and indicator reaction is deducted and does not add the rgb value of image before copper ion, obtain the Δ R of " subtractive image ", Δ G, with Δ B value, the value of three's quadratic sum evolution again and corresponding copper ion concentration of standard solution are built standard concentration curve (as shown in Figure 2).
The mensuration of unknown sample:
Preparation unknown concentration copper ion sample solution, draws 50-100mL testing sample with the syringe with 0.45 ± 0.2 μm of microporous Nylon membrane filtration head, to remove Organic substance in sample, and bulky grain or cotton-shaped impurity；Remove filtering head, transfer the solution in beaker；The pH value regulating post-filtration samples solution with 0.5M dilute hydrochloric acid or sodium hydroxide solution is 6-8.
Take the 12.5 above-mentioned solution of microlitre, join 1mL containing 5 × 10-4The solution of M copper ion indicator, shakes up and is placed in 70 DEG C of thermostat water baths and reacts 30min；
Adopt above-mentioned same fluorescence to take as equipment, the solution after stable is taken as；By its color and standard color comparison card comparison, draw substantially concentration.Extract red, green, blue (RGB) value before and after the change of its color and carry out subtractive, obtain the Δ R of modified-image, Δ G, Δ B value.By Δ R, the value of Δ G, Δ B and the value (13.59) of three's quadratic sum evolution again substitute in copper standard solution curve, by standard curve linear equation (shown in Fig. 2, y=157.62x-0.52), corresponding accurate copper solution concentration (8.95 × 10 to be measured is tried to achieve-5M).
The present invention adopts homemade fluorescence colorimetric equipment, by the ultraviolet light of 365nm specific wavelength, indicator is excited.With electronic imaging device, add the fluorescence color change of front and back indicator as scanner, digital camera etc. catch copper ion.Extract the color changing value of red, green, blue (RGB) three passages in the image of its color change front and back, build copper ion and correspond the standard curve of Concentraton gradient.After color digitized intuitively so that quantitative assay is possibly realized, also improving sensitivity further, the color change that small naked eyes are difficult to see can be displayed more intuitively by the difference after digitized simultaneously.Fluorescence excitation used and image capture device are simple, light are easy to carry about with one, and can be used for on-the-spot test sample, and image acquisition is rapid, it is possible to detect in real time.The method can detect 10-5The ion concentration of M, and can effectively get rid of 9 kinds of ion mutual interferences, there is higher selectivity.
Claims (7)
1. fluorescence equipment is for a colorimetric methods for copper ion detection by quantitative, and including the synthesis of rhodamine quinolines fluorescence indicator, the chelating of copper ion, building of fluorescence colorimetric equipment, fluorescence colorimetric reads, and sample determination, it is characterised in that:
In conjunction with oxine and the rhodamine indicator sequestering power to metal ion, the bright quinolines fluorescence indicator of synthesizing rhodamine, the copper ion of variable concentrations is detected；Adopt fluorescence colorimetric equipment, by digital camera, the change in fluorescence image after copper ion and indicator reaction is acquired, extract the color changing value of red, green, blue (RGB) three passages in the image of its color change front and back, build copper ion and correspond the standard curve of Concentraton gradient；
1) synthesis of rhodamine quinolines indicator: 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: to step 1) the 1mL concentration that obtains is 5 × 10-4The solution of the copper ion fluorescence indicator of M is separately added into the 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 in the water-bath of 70 DEG C ± 2 DEG C after isothermal reaction 30min ± 2min, and the color from light complexion changed of solution becomes redness, and solution tends towards stability；
3) the building of fluorescence colorimetric equipment: select 360-410nm light emitting diode, obtains more single 365nm light source as fluorescent ultraviolet excitation source after filtering via the narrow band pass filter that 365nm light can be allowed to pass through；Taking 200 microlitre steps 2) solution that obtains after reaction is placed in transparent plastic tubule, adopts this excitation source that rhodamine quinolines indicator and itself and the reacted solution of variable concentrations copper ion are excited；
4) fluorescence colorimetric read: via digital imaging device to step 3) excite after solution fluorescence color bottom tubule, carry out imaging, by color by after Photoshop software digital, red (R), green (G), the color changing value of blue (B) three passages extracted before and after color change in image, i.e. R=RAfter reaction-RBefore reaction, G=GAfter reaction-GBefore reaction, B=BAfter reaction-BBefore reaction, build corresponding concentration-gradient curve with copper ion concentration；
5) sample determination: by the copper ion sample of 12.5 μ L unknown concentration and step 1) concentration of rhodamine quinolines copper ion fluorescence indicator that obtains is 5 × 10-4After M solution 1mL reaction, carrying out imaging analysis, the color changing value obtained substitutes into the concentration-gradient curve set up on it, tries to achieve copper ion concentration.
2. method according to claim 1, it is characterised in that: described copper ion indicator is rhodamine quinolines fluorescence indicator, combines rhodamine and the oxine chelation to metal ion.
3. method according to claim 1, it is characterised in that: described digital imaging device is color-image forming apparatus.
4. method according to claim 1, it is characterised in that: indicator is added the reacted color of copper ion and via Photoshop software, can be reflected by digitized form, provides foundation for quantitative analysis.
5. method according to claim 1, it is characterised in that: described plastic syringe is originally cylindrical as water white transparency, and its internal diameter 6mm, external diameter 8mm, high 12mm, light transmission at the bottom of its pipe is good.
6. method according to claim 1, it is characterized in that: described fluorescence excitation equipment is the light emitting diode uviol lamp series connection of 8 4V20mA, and the black that it is fixed on a top seal uniformly overlaps bucket central authorities, set bucket lower ending opening, constitute an area source, it is fixed with the narrow band pass filter that 365nm light only can be allowed to pass through that diameter is 2.5cm below light emitting diode, makes the light passed through only have about 365nm；It addition, be furnished with the bandpass filter of another 400-700nm at the tubule bottom part down putting reaction solution, it is used for filtering out the bias light that LED light lamp sends.
7. the method according to 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, red (R), green (G), the color changing value of blue (B) three passages extracted before and after color change in image, namely after R=R reaction before-R reaction, after G=G reaction before-G reaction, after B=B reaction before-B reaction, substitute into the concentration-gradient curve set up on it, try to achieve copper ion concentration to be measured.
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