CN111323396A - Method for determining laccaic acid A content - Google Patents

Method for determining laccaic acid A content Download PDF

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CN111323396A
CN111323396A CN201811530704.4A CN201811530704A CN111323396A CN 111323396 A CN111323396 A CN 111323396A CN 201811530704 A CN201811530704 A CN 201811530704A CN 111323396 A CN111323396 A CN 111323396A
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Prior art keywords
laccaic acid
solution
acid
concentration
laccaic
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刘兰香
伊冠东
张弘
李坤
张雯雯
李凯
马金菊
徐涓
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Research Institute of Resource Insects of Chinese Academy of Forestry
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Research Institute of Resource Insects of Chinese Academy of Forestry
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence

Abstract

The invention discloses a method for determining laccaic acid A content, which comprises the steps of determining fluorescence intensity of a laccaic acid A-containing sample solution, calculating to obtain the concentration of laccaic acid A in the laccaic acid A-containing sample solution according to a formula Y ═ kX + b, calculating to obtain the laccaic acid A content, wherein Y is the fluorescence intensity, X is the concentration of laccaic acid A in the sample, and k and b are constants－ 7mol/L; the detection result of the laccaic acid A content is accurate and reliable, and the error is less than 4.6%; the operation process is simple in the detection process, a large amount of manpower, material resources and financial resources are saved, the detection cost of the laccaic acid A is reduced, and the method is suitable for wide popularization and application.

Description

Method for determining laccaic acid A content
Technical Field
The invention belongs to the technical field of detection and analysis, relates to a spectral detection method, and particularly relates to a method for determining laccaic acid A content by adopting a fluorescence analysis method.
Background
Lac red pigment is derived from metabolic products of lac worm, is a natural pigment which is allowed to be added into food, and is widely applied to dyeing of cotton, wool, textiles and the like. The lac dye has the main dyeing component of lac acid mixture, mainly contains 5 components of A, B, C, D, E, etc., wherein the lac acid A accounts for about 85% of the total mass, and the lac acid A all belong to anthraquinone compounds, and the structural formula of the lac dye is shown as a formula (1). Anthraquinone and its derivatives can emit fluorescence due to their good planar and conjugated structures, and are widely used to identify various metal ions or other anions in different systems. Lac red pigment has been shown to be fluorescent, however, the fluorescent properties of chemically defined single-component laccaic acids have not been reported.
Figure RE-GDA0001954427210000011
Compared with synthetic pigments, natural pigments have the advantages of high safety, physiological activity and the like, and are more and more popular with consumers in recent years, so that strict requirements are also made on safe use and detection of the natural pigments in food, medicine and other related industries. For example, the lac dye is proved to be safe through strict toxicological experiments and safety evaluation, but the national standard still clearly defines that the maximum dosage of the lac dye added into food such as drinks, cakes, seasonings and the like does not exceed 0.5 g/kg. At present, the common detection methods of natural pigments mainly include High Performance Liquid Chromatography (HPLC), high performance liquid chromatography-mass spectrometry (HPLC-MS), Capillary Electrophoresis (CE), Thin Layer Chromatography (TLC), and the like, but the related reports of the detection method of lac red pigments are few, and no related report of the fluorescence method for detecting the lac red pigments or lac acid a is available. Therefore, in order to meet the requirements of the safety development of foods and medicines, the establishment of the rapid, accurate and sensitive detection of the lac pigment or the lac acid A in the foods has practical significance. The laccaic acid A is the most main effective component in the lac red pigment and is also an important index for evaluating the quality of the lac red pigment, so that the development of the lac red acid A with a determined chemical structure has important significance for qualitative and quantitative detection.
Disclosure of Invention
The invention aims to provide a method for measuring laccaic acid A aiming at the technical defects in the existing method for detecting laccaic acid A.
In order to achieve the object of the present invention, in one aspect, the present invention provides a method for determining the amount of laccaic acid a, comprising determining the fluorescence intensity of a sample solution containing laccaic acid a; then, calculating according to a formula (1) to obtain the concentration of the laccaic acid A in the laccaic acid A-containing sample solution; and then calculating the content of the laccaic acid A, wherein the formula (1) is as follows:
Y＝kX+b (1)
wherein Y is the fluorescence intensity, a.u; x is the concentration of laccaic acid A in the sample, mol/L; k. b is a constant.
The laccaic acid A-containing sample solution is prepared by the following method: dissolving a sample containing the laccaic acid A in an ethanol-water solution to prepare a laccaic acid A sample solution with a certain concentration.
In particular, the ethanol-water solution is ethanol-water solution with the volume percentage concentration of 25-100%, preferably ethanol-water solution with the volume percentage concentration of 75-100%, and more preferably ethanol-water solution with the volume percentage concentration of 75%.
In particular, the concentration of the laccaic acid a sample solution was 2.0 × 10-6-4.0×10-3mol/L, preferably 1.0 × 10-5-8.0×10-5mol/L。
Particularly, the method also comprises the steps of adding an accelerant into the laccaic acid A sample solution, uniformly mixing, and then measuring the fluorescence intensity of the solution.
Wherein the accelerator is Mg2+、Zn2+Or Al3+Preferably Al3+The solution of (1).
In particular, Mg in the accelerator solution2+、Zn2+Or Al3+The concentration of (B) is 0.0001 to 0.08mol/L, preferably 0.04 mol/L.
In particular, the volume ratio of the added accelerator solution to the laccaic acid a sample solution is 1: 4-6, preferably 1: 5.
In particular, the measurement conditions for measuring the fluorescence intensity of the laccaic acid A-containing sample solution are as follows: the excitation wavelength (Ex) was 380 nm.
In particular, the determination conditions for determining the fluorescence intensity of the laccaic acid A-containing sample solution are as follows: the excitation wavelength (Ex) was 380nm, the slit width was 10nm, the scanning rate was 12000nm/min, and the photomultiplier voltage was 950V.
The invention also provides a method for determining the content of laccaic acid A, which comprises the following steps:
1) preparing accelerator solution
Adding soluble Mg2+Or Zn2+Or Al3+Dissolving salt in water to prepare an accelerator aqueous solution;
2) preparing laccaic acid A sample solution to be detected
Dissolving a sample to be detected containing the laccaic acid A in an ethanol-water solution to prepare a laccaic acid A sample solution to be detected;
3) uniformly mixing the laccaic acid A sample solution to be detected with an accelerator solution, and then measuring the fluorescence intensity of the mixed solution;
4) calculating the concentration of the laccaic acid A in the laccaic acid A sample solution to be detected according to the formula (1); and then calculating to obtain the laccaic acid A content of the laccaic acid A in the sample to be detected containing the laccaic acid A, wherein the formula (1) is as follows:
Y＝kX+b (1)
wherein Y is the fluorescence intensity, a.u; x is the concentration of laccaic acid A in the sample, mol/L; k. b is a constant.
Wherein Mg in the accelerator solution prepared in the step 1)2+Or Zn2+Or Al3+The concentration of (B) is 0.0001 to 0.08mol/L, preferably 0.04 mol/L.
In particular, the metal salt is selected from MgCl2。6H2O、ZnCl2、AlCl3、AlCl3.6H2O、Al(NO3)3、 Al2(SO4)3Or alum, preferably AlCl3、AlCl3.6H2O。
Wherein, the volume percentage concentration of the ethanol-water solution in the step 2) is 25-100%, preferably 75-100%, and more preferably 75%.
Particularly, the concentration of the laccaic acid A sample solution to be detected is 4.0 × 10-3-2.0×10-6mol/L, preferably 1.0 × 10-5-8.0×10-5mol/L。。
Wherein, the volume ratio of the laccaic acid A sample solution to be detected to the accelerator solution in the step 3) is 4-6: 1, preferably 5: 1.
In particular, in step 4), formula (1) is specifically 326.2X + 467.7.
Compared with the prior art, the method has the following advantages and benefits:
1. the method has simple sample pretreatment, the laccaic acid A sample is dissolved in the mixed solvent of ethanol and water to prepare a solution, and the method does not need complicated process means and is quick, simple and convenient;
2. the method for detecting the content of the laccaic acid A is simple to operate, and can detect the laccaic acid A by using a fluorimeter;
3. in the detection process of the content of the laccaic acid A, the accelerator is added to enhance the fluorescence intensity of the laccaic acid A sample solution, the detection sensitivity is obviously improved, and the minimum detection limit of the laccaic acid A is 2.0 × 10-7mol/L；
4. In the process of detecting the content of the laccaic acid A, the accelerant is added, the content of the laccaic acid A is measured through the working curve, the detection result is accurate and reliable, and the error is less than 4.6%.
5. The method does not need a highly professional technician to operate, saves a large amount of manpower, material resources and financial resources, and reduces the detection cost.
Drawings
FIG. 1A is a graph of the effect of different solvents on the fluorescence spectra of laccaic acid A, wherein: a-water, b-methanol, c-ethanol, d-n-butanol, e-DMF, f-DMSO;
FIG. 1B is a graph of the effect of different solvents on the absorbance spectrum of laccaic acid A, wherein: a-water, b-methanol, c-ethanol, d-n-butanol, e-DMF, f-DMSO;
FIG. 2 is a graph of the effect of ethanol/water solutions of different ethanol volume fractions on the fluorescence spectra of laccaic acid A, wherein: a-water; b-25% (v/v) ethanol/water mixture; c-50% (v/v) ethanol/water mixture; d-75% (v/v) ethanol/water mixture; e-90% (v/v) ethanol/water mixture; f-absolute ethyl alcohol;
FIG. 3 is a graph showing the effect of concentration on fluorescence intensity and emission wavelength of laccaic acid A, wherein a is fluorescence intensity and b is emission wavelength;
FIG. 4A is a graph of the effect of pH on the fluorescence intensity and emission wavelength of laccaic acid A, where a is the emission wavelength; b is fluorescence intensity;
FIG. 4B is a graph of the effect of pH on the absorbance spectrum of laccaic acid A;
FIG. 5 is a graph showing the effect of different metal ions on the fluorescence intensity of laccaic acid A;
FIG. 6 shows Zn2+Or Al3+A graph of the effect of the concentration of (a) on the fluorescence intensity of laccaic acid A;
FIG. 7A shows the addition of Zn to laccaic acid A2+Or Al3+A fluorescence spectrum of (a);
FIG. 7B shows the addition of Zn to laccaic acid A2+Or Al3+A visible light spectrum of (a);
FIG. 8 shows the addition of Al to laccaic acid A by other metal ions3+Influence graph of fluorescence intensity of (a);
FIG. 9 is a graph showing the relationship between the concentration of laccaic acid A and the fluorescence intensity;
FIG. 10 shows Al3+Working curve graph of concentration of laccaic acid A and fluorescence intensity in the presence of laccaic acid A.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
F-4600 fluorescence spectrometer (HITACHI, Japan); carrier Series UV-visible absorption Spectroscopy (Agilent Technologies, USA); WD-9403C UV instrument (Beijing, six Biotech Co., Ltd.); an AB 204-type precision electronic balance (mettler-toledo (china)); CPC-505 desktop pH meter (European ELMETRON Corp.); TY742X2A type water purification machine (Barnstead, usa).
The lac haematochrome and lac acid A (more than or equal to 95 percent) are provided by resource insect research institute of China forestry scientific research institute; hydrochloric acid, sodium hydroxide, methanol, ethanol, N-butanol, N, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), metal inorganic salts except AgNO3Except that the rest are chloride salts, Guangdong Guanghua science and technology Co., Ltd (the above reagents are all analytically pure); the water is deionized water.
Test example 1 selection of solvent type
Accurately weighing 6 parts of laccaic acid A with weight of 2.7mg respectively, dissolving in 25.0mL water, methanol, ethanol, n-butanol, DMF, and DMSO respectively to obtain a solution with concentration of 2 × 10-4Performing ultrasonic treatment on a mol/L laccaic acid A solution at room temperature (20-35 ℃) for 10min, and performing spectral measurement by respectively adopting an F-4600 fluorescence spectrometer and a Cary Series ultraviolet-visible absorption spectrometer, wherein:
when in fluorescence spectrum measurement, the excitation wavelength is set to be 380nm, the slit width is 10nm, the scanning speed is 12000nm/min, and the voltage of the photomultiplier is 950V; the scanning speed in the process of scanning the full wavelength of the ultraviolet-visible absorption spectrum is 600 nm/min. The measurement results are shown in FIGS. 1A and 1B.
As shown in fig. 1A, the fluorescence intensities of laccaic acid a in different solvents are in the order of small to large: water < methanol < DMSO < n-butanol < ethanol < DMF. Furthermore, the maximum emission wavelength (Em) of laccaic acid a is solvent dependent, with Em lying around 597nm in water and alcohol and around 630nm in DMF and DMSO. As shown in FIG. 1B, the maximum absorption wavelength (. lamda.max) of laccaic acid A in the visible region was changed with changes in fluorescence intensity and Em, and the minimum was 488nm in water; the largest is 501nm in DMF. Experimental results show that different solvents have important influence on the fluorescence emission wavelength and intensity of the laccaic acid A and the absorbance of the laccaic acid A in a visible light region. The reason for this is that water and alcohol are protic solvents, which easily form hydrogen bonds with the chromophoric acid molecules, which contribute to the aggregation of chromophoric acid molecules, whereas water is the most polar of all solvents studied, and the hydrophilic action of chromophoric acid molecules is sufficient to overcome the electrostatic repulsion between chromophoric acid anions, thereby reducing the fluorescence thereof, and therefore, the fluorescence intensity of laccaic acid a in water is minimal. DMSO and DMF belong to aprotic solvents, which are easily solvated with solute chromophoric acid molecules, and since S ═ O or C ═ O bonds exist in the solvent, they contribute to pi → pi × -transition when fluorescence of the chromophoric acid molecules is excited, and N atoms in DMF have lone pair electrons, enhancing this effect, so that fluorescence of laccaic acid a is the strongest in DMF. It can be seen that the fluorescence intensity of laccaic acid a is mainly influenced by the chemical properties of the solvent itself, and secondly, the polarity of the solvent is also influenced to some extent. The laccaic acid A has very low solubility in water and weak fluorescence; the solubility in ethanol is high, and the fluorescence is strong; the organic solvents such as DMSO, DMF and the like have certain toxicity, so that ethanol is a better solvent for laccaic acid A, but in consideration of more water system environment in practical application, in order to avoid great change of system environment caused by direct mixing of water and ethanol, an ethanol/water mixed system is selected as a suitable solvent.
Experimental example 2 selection of solvent ethanol and Water
Mixing absolute ethanol with water to prepare ethanol-water solution, wherein the ethanol-water solution is controlled by 25 percent, 50 percent, 75 percent, 90 percent and 100 percent of ethanol by volume respectively.
Weighing 5.4mg laccaic acid A, dissolving in 50.0mL ethanol-water solution with different volume fractions to obtain a solution with a concentration of 2 × 10-4Respectively measuring the concentration of laccaic acid A-ethanol-water solution by F-4600 fluorescence spectrometerThe fluorescence intensity and the measurement result are shown in FIG. 2.
As shown in fig. 2: with the increase of the volume fraction of the ethanol, the fluorescence intensity of the solution is obviously increased and the emission wavelength is red-shifted, so that the solution with higher volume fraction of the ethanol is preferably selected when the fluorescence property of the laccaic acid A is inspected; when the fluorescence property of the laccaic acid A is considered, the volume fraction of ethanol in the ethanol/water mixed solution is 25-100%, preferably 75%.
Test example 3 influence of concentration of laccaic acid A on fluorescence intensity and emission wavelength
Weighing 107.5mg laccaic acid A, dissolving in 50.0mL 75% ethanol-water solution to obtain a solution with a concentration of 4 × 10-3Adding 75% ethanol-water solution into the stock solution of laccaic acid A at mol/L, and gradually diluting to obtain a solution with a concentration of 2.0 × 10-6、2.0×10-5、4.0×10-5、1.0×10-4、2.0×10-4、5.0×10-4、1.0×10-3、2.0×10-3The sample solution to be tested in mol/L is reserved, an F-4600 fluorescence spectrometer is adopted for spectrum measurement, the fluorescence intensity of the laccaic acid A-ethanol/water solution is measured, the influence of the concentration on the laccaic acid A spectrum property is inspected, and the measurement result is shown in figure 3.
As shown in FIG. 3, when the concentration of laccaic acid A is 2.0 × 10-6～2.0×10-4In the mol/L range, the fluorescence intensity of laccaic acid A (the curve of a is fluorescence intensity) and Em (b represents emission wavelength) both increase with increasing concentration, and when the concentration is 2.0 × 10-4The fluorescence is strongest when mol/L, and Em is increased from 585nm to 598 nm; the fluorescence intensity of the system will decrease sharply until quenching, with the Em remaining substantially at around 600nm, as the concentration of laccaic acid A continues to increase. This is probably because the fluorescent molecules aggregate to form dimers or multimers in a low concentration range to contribute to fluorescence enhancement, but when the concentration of the fluorescent molecules increases to a certain extent, the probability that molecules in an excited state collide with molecules in a ground state to deactivate the molecules in the excited state increases, and the fluorescent molecules undergo self-energy absorption to cause fluorescence reduction or even quenching.
Test example 4 influence of pH value of solution on fluorescence and ultraviolet-visible spectrum of laccaic acid A
Stock solutions of 0.1mol/L HCl and 0.1mol/L NaOH were prepared, respectively, and then diluted appropriately to pH 1-13, respectively, and corrected with a pH meter.
Respectively weighing 13 parts by weight of laccaic acid A2.7 mg, respectively dissolving in 25.0mL of the above HCl and NaOH diluents with pH3-13, and making into the solution with concentration of 2.0 × 10-4Performing ultrasonic treatment on mol/L laccaic acid A solution at room temperature for 10min, performing spectral measurement by respectively using an F-4600 fluorescence spectrometer and an ultraviolet-visible absorption spectrometer (Cary Series), and investigating the influence of HCl and NaOH solutions with different pH values on the spectral properties of the laccaic acid A, wherein the measurement results are shown in FIGS. 4A and 4B.
When the pH of the solution is less than or equal to 3, the solubility of the laccaic acid A is very low and is less than 2.0 × 10-4mol/L (0.1mg/mL), resulting in a very low fluorescence intensity.
As shown in FIGS. 4A and 4B, when the pH of the solution is between 4 and 10, the fluorescence intensity of laccaic acid A is slightly influenced by the pH, almost remains unchanged, and the absorption spectrum has no obvious difference. When the solution pH reached 11, the color of the solution changed to purple, the fluorescence was almost quenched, and a flat and broad absorption peak appeared in the visible spectrum (see FIG. 4B). When the pH value is more than 11, the fluorescence intensity of the laccaic acid A is rapidly and remarkably increased, double absorption peaks appear in a visible light region, and the maximum absorption wavelengths are respectively 530nm and 565nm (as shown in figure 4B).
The difference of fluorescence intensity changes of laccaic acid A in different pH ranges is possibly related to ionization of carboxyl and hydroxyl (phenol) in the molecular structure of the laccaic acid A, the pH is 4-10, and the laccaic acid A mainly exists in a molecular form; the pH value is about 11, and two carboxyl groups in molecules are ionized; when the pH is > 11, the hydroxyl groups (phenols) begin to ionize and laccaic acid A exists predominantly in the ionic form. Therefore, when the influence of other factors on the fluorescence of the laccaic acid A is considered, a solution with the pH value of 4-10 can be selected.
Test example 5 Effect of different Metal ions on fluorescence intensity of Lactocrylic acid A
Weighing 2.7mg laccaic acid A, dissolving in 25.0mL 75% ethanol-water solution, performing ultrasonic treatment at room temperature for 10min to obtain laccaic acid A with concentration of 2 × 10-4And (3) a laccaic acid A standard solution in mol/L.
Respectively taking 5.0mL of laccaic acid A solution and 1.0mL of metal ions (4 × 10)-3mol/L) solution is mixed in a centrifuge tube, the mixture is kept stand for 20min after being mixed evenly, then the fluorescence intensity is measured, and the fluorescence influence of metal ions on laccaic acid A is inspected, wherein the metal ions are Na+、K+、Mg2+、Ca2+、Cu2+、Zn2+、Cd2+、Co2+、 Fe2+、Fe3+、Ag+、Cr3+、Ba2+、Al3+、NH4 +、Mn2+、Ni2+、Pb2+And Hg2+. The measurement results are shown in FIG. 5:
as shown in FIG. 5, Cu is in 75% ethanol-water solution2+、Fe3+、Fe2+And Pb2+All four ions quench the fluorescence of laccaic acid a almost completely; na (Na)+、K+、Cd2+、Ag+、NH4 +、Mn2+And Hg2+The plasma metal ions hardly have influence on the fluorescence intensity; mg (magnesium)2+Slightly increase the fluorescence, Zn2+And Al3+But the fluorescence intensity of the laccaic acid A is obviously enhanced. The reason may be that phenolic hydroxyl and carboxyl in the molecule of laccaic acid a are good ligands of metal ions, and are easy to form complexes with the metal ions, so that the functions of electron transfer, energy transfer and the like occur, and meanwhile, under the influence of extra-nuclear electron arrangement and charges of the metal ions, the effects of different metal ions on fluorescence generation of laccaic acid a are different.
Test example 5A Zn2+Or Al3+Influence on fluorescence intensity of laccaic acid A
Zn with the concentration of 0.1mol/L is prepared2+Or Al3+Aqueous solution, and gradually diluted with water to various concentrations (8.0 × 10)－2～1.0×10－4mol/L) and 5.0mL of 2.0 × 10－4The 75 percent ethanol water solution of the laccaic acid A in mol/L is respectively mixed with Zn in 1.0mL of different concentrations2+Or Al3+The diluted solution is mixed uniformly, fluorescence is detected, and Zn is inspected2 +Or Al3+The relationship between the concentration and the fluorescence intensity of laccaic acid A is shown in FIG. 6.
5.0mL of 2.0 × 10－4mol/L of laccaic acid A in 75% ethanol aqueous solution and 1.0mLZn2+(4×10－3mol/L) or 1.0mlAl3+(2.0×10－2mol/L) are mixed evenly, then the absorbance value is detected, and Zn is inspected2+Or Al3+The results of measurement are shown in FIGS. 7A and 7B in relation to the absorption spectrum of laccaic acid A.
As shown in FIG. 6, when Zn2+Is less than 4 × 10－3At mol/L, the fluorescence intensity of laccaic acid A is dependent on Zn2+But continues to increase Zn2+The concentration of (b) is such that insoluble material begins to form in the solution and the fluorescence gradually decreases until complete quenching. With addition of Zn2+In contrast to when Al is present3+Is less than 4.0 × 10－2At mol/L, the fluorescence intensity of laccaic acid A is dependent on Al3+The concentration is increased obviously, and the fluorescence intensity of the laccaic acid A is almost kept unchanged when the concentration is increased continuously. When Al is present3+When the concentration is more than 0.04mol/L, the fluorescence intensity of laccaic acid A is dependent on Al3+The increase in concentration hardly changed. Thus, the mechanism of the enhancement of the fluorescence of laccaic acid A by the two may be different, and the inference can be further verified by the fluorescence spectrum and the ultraviolet-visible spectrum.
As shown in FIGS. 7A and 7B, Zn is added to the laccaic acid A solution2+When it is used, the emission wavelength and the maximum absorption wavelength in the visible region are not substantially changed, however, Al is added3+When the emission wavelength of the laccaic acid solution is shifted from 590nm to about 625nm, and simultaneously, the visible spectrum shifts from 488nm to the long wave direction and is split into two peaks, namely 522nm and 562 nm. From this, Zn can be estimated2+The reason for enhancing the fluorescence of laccaic acid A may be Zn2+The photo-induced electron transfer occurs under the action of N atom and-OH in the molecule of the chromoacid, and Al3+Forms different complexes with-OH and-COOH at a plurality of sites in a chromophoric acid molecule, and generates fluorescence energy resonance transfer. Taken together, among the metal ions examined, only Al3+The specificity of significantly enhancing the fluorescence intensity of laccaic acid a appeared.
Experimental example 6 other Metal ion Pair Al3+Influence of the fluorescence intensity of Lactocrylic acid A
Weighing laccaic acid A, dissolving in 75% ethanol-water solution to obtain a solution with a concentration of 2 × 10-4A laccaic acid A solution of mol/L; respectively measuring 5.0mL laccaic acid A solution, and mixing with 1.0mL LAl3+(4×10－3mol/L) of the aqueous solution are mixed in a centrifuge tube, and then 1.0mL of 4.0 × 10 is added－3mol/L of other metal ions (Na)+、K+、Ca2+、Cu2+、Cd2+、Co2+、Fe2+、Fe3+、Ag+、Cr3+、Ba2+、NH4 +、 Mn2+、Ni2+、Pb2+And Hg2+) And (4) uniformly mixing the solution, and measuring the fluorescence intensity of the solution. The measurement results are shown in FIG. 8.
As shown in FIG. 8, in the case of Al3+Adding other metal ions, Fe into 75% ethanol water solution3+With Al3+Quenching of fluorescence of the system in coexistence, Zn2+、Pb2+With Al3+The fluorescence intensity of the system is reduced when the metal ions coexist, and the fluorescence intensity of the system is not obviously changed when other metal ions coexist. The results show that in the absence of Fe3+、Zn2+And Pb2+In ionic environment, laccaic acid A is coupled to Al3+The anti-interference performance of selective recognition is stronger, and the selectivity is better.
Test example 7 Al3+Relationship between concentration of laccaic acid A and fluorescence intensity thereof in the presence of laccaic acid A
Al with the concentration of 0.04mol/L is prepared3+The aqueous solution is reserved;
dissolving laccaic acid A in 75% ethanol-water solution to obtain a solution with a concentration of 4.0 × 10－3The stock solution of mol/L is diluted by 75 percent ethanol-water solution to different concentrations (2.0 × 10)－3～1.0×10－6mol/L) as a sample to be detected for standby;
taking 5.0mL of laccaic acid A solution with different concentrations in a centrifuge tubeAdding 1.0ml of lal respectively3+The fluorescence intensity of the aqueous solution is measured after the aqueous solution is uniformly mixed, and the measurement result is shown in figure 9; while adding no 1.0ml of LAl3+The aqueous solution is used as a control, the fluorescence intensity of the laccaic acid A solution with different concentrations is measured, and the measurement result is shown in figure 9;
the concentration of laccaic acid A is plotted on Al by using the abscissa as the concentration of laccaic acid A and the ordinate as the fluorescence intensity3+Operating curves in the presence, as in fig. 10.
As shown in FIG. 9, in 75% ethanol solution of laccaic acid A, regardless of whether Al is added to the solution3+The fluorescence intensity of laccaic acid A shows the change of increasing first and then decreasing with the increase of the concentration. However, in the absence of Al3+When the concentration of laccaic acid A is detected by fluorescence method, the error is large, and Al is added3+Then the fluorescence intensity can be enhanced by more than 6 times at most. Therefore, Al can be examined3+The fluorescence intensity of laccaic acid a in the presence of the compound is related to its concentration.
In addition, as can be seen from FIG. 6, when Al is added3+When the concentration is more than 0.04mol/L, the fluorescence intensity of laccaic acid A is dependent on Al3+The concentration increase hardly changed any more, so 0.04mol/L of Al was selected3+Tests were carried out. The results show that laccaic acid A is in Al3+(0.04mol/L) in the presence of 1.0 × 10－5～8.0×10－5The concentration range of mol/L has good linear relation with the fluorescence intensity, and the linearity is fitted to obtain a working curve equation: y 326.2x +467.7, coefficient of linear correlation R20.9952, as in fig. 10. Thus, in Al3+And (0.04mol/L) detecting the content of the laccaic acid A in the sample by a fluorescence method.
Test example 8 lowest detection Limit test
Accurately weighing 10.7mg laccaic acid A, dissolving in 100mL 75% ethanol water solution to obtain a solution with a concentration of 2.0 × 10－4The stock solution of mol/L is diluted to 2.0 × 10 step by step－6、1.0×10－6、2.0×10－7Taking mol/L as a sample to be detected for later use;
241.4mgAlCl is accurately weighed3.6H2Dissolving O in 25mL of ultrapure water to prepare 0.04mol/L Al3+Aqueous solution
Respectively mixing 5mL of sample solution to be detected with 1mL of sample solution3+The concentration of laccaic acid A corresponding to the fluorescence intensity of 3 times of instrument background signal generated by matrix blank is the minimum detection limit concentration, and the minimum detection limit concentration of laccaic acid A in the method is 2.0 × 10－7mol/L。
Example 1
Commercially available orange flavored beverages: the concentration of laccaic acid A contained in the composition is as follows: 0 mol/L;
preparing AlCl with the concentration of 0.04mol/L3Accelerator aqueous solution for standby;
3 parts of laccaic acid A (20.0mg) were weighed separately and added to 10mL of a known brand orange-flavored drink A to prepare 3.7 × 10－3The stock solution of mol/L is then diluted with 75% ethanol solution to a concentration of 1.5 × 10-5、3.2×10-5、5.5×10-5Taking mol/L as a sample solution to be detected for later use;
taking 5.0mL of sample to be detected, adding 1.0mL of LAl into a centrifuge tube respectively3+The fluorescence intensity of the aqueous solution is measured after the aqueous solution is uniformly mixed, the obtained data is substituted into a working curve equation, and the concentration of the laccaic acid A in the sample to be measured is calculated;
the accuracy of the established standard equation was verified by calculating the sample recovery rate, which is × 100% of the calculated concentration of laccaic acid a/the actual added concentration of laccaic acid a in the beverage.
The measurement results are shown in Table 1.
Example 2
And B, market selling of the lemon flavor beverage: the concentration of laccaic acid A contained in the composition is as follows: 0 mol/L;
preparing AlCl with the concentration of 0.04mol/L3Accelerator aqueous solution for standby;
3 parts of laccaic acid A (20.0mg) were weighed out separately and added to 10mL of lemon-flavored beverage B to prepare 3.7 × 10－3The stock solution of mol/L is diluted by 75 percent ethanol water solutionReleased to different concentrations of 1.5 × 10-5、 3.2×10-5、4.5×10-5The mol/L of sample solution to be detected is reserved;
taking 5.0mL of sample to be detected, adding 1.0mL of LAl into a centrifuge tube respectively3+The fluorescence intensity of the aqueous solution is measured after the aqueous solution is uniformly mixed, the obtained data is substituted into a working curve equation, and the concentration of the laccaic acid A in the sample to be measured is calculated;
the accuracy of the established standard equation was verified by calculating the sample recovery rate, which is × 100% of the calculated concentration of laccaic acid a/the actual added concentration of laccaic acid a in the beverage.
The measurement results are shown in Table 1.
Table 1 laccaic acid a test application experimental results (n ═ 3)
Figure RE-GDA0001954427210000121
Theoretical values represent the concentration of laccaic acid a added to the beverage.
As shown in the experimental results of Table 1, Al is used3+The method is characterized in that the method is used as a standard solution, the content of laccaic acid A added in two different beverages is detected by a fluorescence method, the sample recovery rate is 95.0-109.3%, and the relative standard deviation is 1.9-5.4%, and the result shows that the method has the advantages that the content of laccaic acid A added in Al in the two different beverages is 95.0-109.3%, and the Al content is higher than3+Under the condition of existence of (0.04mol/L), the content of the laccaic acid A in the sample can be detected by a fluorescence method, and the method has practical application value.
The method has the advantages of simple operation, small error, high sensitivity and accurate and reliable result, and provides an effective method for rapidly detecting the content of the laccaic acid A in food and other samples.
Example 3
Weighing 27.0mg laccaic acid A, dissolving in 25.0mL 75% ethanol-water solution to obtain a solution with a concentration of 2.0 × 10-3A laccaic acid A stock solution of mol/L;
adding 75% ethanol-water solution into the stock solution, and gradually diluting to obtain solution with concentration of 2.0 × 10-5、4.0×10-5、6.0×10-5、8.0×10-5The mol/L of sample solution to be detected is reserved;
respectively taking 5mL of sample solution to be detected, and then adding 1mL of AlCl with the concentration of 0.04mol/L into each sample solution to be detected3The promoter aqueous solution is subjected to F-4600 fluorescence spectrometer to measure the fluorescence intensity of the laccaic acid A-ethanol/aqueous solution;
the measured fluorescence intensity was calculated by substituting the working curve equation of y 326.2x +467.7 to obtain the concentration of laccaic acid A of 2.09 × 10-5、4.17×10-5、6.16×10-5、7.81×10-5And mol/L, comparing with the concentration of the corresponding sample solution to be detected, wherein the error is less than 4.6 percent, and the determination result is consistent with the actual concentration.
The invention firstly proposes to add Al into the laccaic acid A3+(0.04mol/L) to quantitatively detect laccaic acid A, and the fluorescence intensity of the system is found to have a good linear relation with the concentration thereof, and the fitting equation is that y is 326.2x +467.7, R2The fluorescence analysis method for detecting laccaic acid A content is successfully established at 0.995, and the fluorescence analysis method for laccaic acid A established by the method of the invention can be set at 1.0 × 10-5-8.0×10-5mol.L-1The method has the advantages of simple and convenient operation, small error, high sensitivity and accurate and reliable result, and provides an effective method for rapidly detecting the content of the laccaic acid A in food and other samples.

Claims (10)

1. A method for determining the content of laccaic acid A is characterized by comprising the steps of determining the fluorescence intensity of a laccaic acid A-containing sample solution; then, calculating according to a formula (1) to obtain the concentration of the laccaic acid A in the laccaic acid A-containing sample solution; and then calculating the content of the laccaic acid A, wherein the formula (1) is as follows:
Y＝kX+b (1)
wherein Y is the fluorescence intensity, a.u; x is the concentration of laccaic acid A in the sample, mol/L; k. b is a constant.
2. The method of claim 1, wherein the laccaic acid a-containing sample solution is prepared by the following method: and dissolving the laccaic acid A-containing sample in an ethanol-water solution to prepare a laccaic acid A sample solution.
3. The method according to claim 2, characterized in that the solvent is selected from the group consisting of ethanol-water solutions with a concentration of 25-100% by volume, preferably from 75-100% by volume, more preferably from 75% by volume.
4. The method of claim 1, further comprising adding an accelerator to the laccaic acid a sample solution, and after mixing, measuring the fluorescence intensity of the solution.
5. The method of claim 4, wherein the promoter is Mg2+、Zn2+Or Al3+Preferably Al3 +The solution of (1).
6. The method of claim 5, wherein Mg is present in the accelerator solution2+、Zn2+Or Al3+The concentration of (B) is 0.0001 to 0.08mol/L, preferably 0.04 mol/L.
7. The method of claim 4, wherein the ratio of the volume of the accelerator solution added to the laccaic acid A sample solution is 1: 4-6, preferably 1: 5.
8. The method of claim 1, wherein the conditions for measuring the fluorescence intensity of the laccaic acid a-containing sample solution are as follows: the excitation wavelength (Ex) was 380 nm.
9. A method for measuring the content of laccaic acid A is characterized by comprising the following steps:
1) preparing accelerator solution
Adding soluble Mg2+Or Zn2+Or Al3+Dissolving salt in water to prepare an accelerator solution;
2) preparing laccaic acid A sample solution to be detected
Dissolving a sample to be detected containing the laccaic acid A in an ethanol-water solution to prepare a laccaic acid A sample solution to be detected;
3) uniformly mixing the laccaic acid A sample solution to be detected with an accelerator solution, and then measuring the fluorescence intensity of the mixed solution;
4) calculating the concentration of the laccaic acid A in the laccaic acid A sample solution to be detected according to the formula (1); and then calculating to obtain the laccaic acid A content of the laccaic acid A in the sample to be detected containing the laccaic acid A, wherein the formula (1) is as follows:
Y＝kX+b (1)
wherein Y is the fluorescence intensity, a.u; x is the concentration of laccaic acid A in the sample, mol/L; k. b is a constant.
10. The method for determining the laccaic acid a content of claim 9, wherein said formula (1) in step 4) is 326.2X + 467.7.
CN201811530704.4A 2018-12-14 2018-12-14 Method for determining laccaic acid A content Pending CN111323396A (en)

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