CN107389572B - Method for detecting copper chlorophyll in olive oil - Google Patents
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Abstract
A method for detecting copper chlorophyll in olive oil is characterized by comprising the following steps: firstly, selecting primary soybean oil with characteristic absorption wavelength of 360-860nm, and preparing a standard stock solution of copper chlorophyll by using the primary soybean oil as a diluent; secondly, preparing standard use solution of copper chlorophyll from blank olive oil without copper chlorophyll; thirdly, detecting the sample by adopting a spectrophotometer, and taking the absorbance of one wavelength or the average value of the absorbance of more than two wavelengths of 400-440nm or 620-660nm as the characteristic absorbance of the copper chlorophyll in the sample; fourthly, correcting the absorbance by using one or more wavelengths of 360-400nm, 440-540nm and 660-800 nm; and fifthly, quantifying by adopting an external standard method to obtain the content of the copper chlorophyll. The method has the advantages of intuition, rapidness, convenience, maturity, low price and the like.
Description
Technical Field
The invention relates to the technical field of illegal additive detection in olive oil, in particular to a method for detecting copper chlorophyll in olive oil.
Background
Chlorophyll derivatives such as copper chlorophyll, sodium copper chlorophyll and potassium copper chlorophyll are widely used in the food industry as colorants. Copper chlorophyllin (copperchlorophyllil), CNS number 08.153, INS number 141i, are fat-soluble compounds, and sodium copper chlorophyllin specified in national standard GB2760-2014 "national standard for food additives for food safety" can be used as colorant for cream, candy, and baked food. Sodium copper chlorophyllin and potassium copper chlorophyllin (chlorophyllin copper chlorophyllins, sodium and copper chlorophyllins) CNS No. 08.009 and INS No. 141ii are water-soluble compounds, and the sodium copper chlorophyllin and the potassium copper chlorophyllin specified in national standard GB2760-2014 (national standard for food additives for food safety) can be used as coloring agents for foods such as frozen drinks, canned vegetables, fruit and vegetable juice, compound wine and the like. Copper chlorophyllin, sodium copper chlorophyllin and potassium copper chlorophyllin can not be used in edible vegetable oil.
The olive oil is an ideal edible oil in new century gradually due to its unique physicochemical index and health-care function, is rich in monounsaturated fatty acids, namely oleic acid, linoleic acid and linolenic acid, as well as vitamin A, vitamin B, vitamin D, vitamin E, vitamin K, antioxidant and the like, and is considered to be the oil which is most suitable for human nutrition in the oil discovered so far. Since many of the underrefined virgin olive oils sold in the market contain partial chlorophyll and look yellow-green, people can regard the yellow-green olive oil as the pure olive oil, which also becomes an important basis for people to select the olive oil.
Therefore, some merchants forbid the ban of the prohibition of the participation of the soybean oil and other cheap oil and fat by the prohibition of the prohibition. The fake olive oil prepared by adding copper chlorophyll, sodium copper chlorophyll and potassium copper chlorophyll into cheap grease such as soybean oil has the disadvantages of damaging economic benefits of consumers, and causing damage to liver and kidney of human body or anemia after long-term consumption. Therefore, detection of illegal additives such as copper chlorophyll, sodium copper chlorophyll and potassium copper chlorophyll in oils and fats is imminent.
At present, in domestic and foreign literatures and records of detection standards, spectrophotometry methods such as "spectrophotometry for measuring sodium copper chlorophyll in food GB/T23749-2009" and "method for detecting sodium copper chlorophyll in olive oil" in chinese patent (application No. 201610465792.9) are available; the liquid phase method comprises the following steps: chinese patent "a method for measuring sodium copper chlorophyllin in olive oil" (application number: 201410229192.3); liquid chromatography tandem mass spectrometry such as: chinese patent "detection method for detecting sodium copper chlorophyllin in olive oil by liquid chromatography tandem mass spectrometry (LC-MS/MS)" (application number: 201310629900.8). No method for detecting copper chlorophyll in olive oil is available.
Disclosure of Invention
The purpose of the invention is: the method for detecting the copper chlorophyll in the olive oil is easy to operate and good in accuracy.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for detecting copper chlorophyll in olive oil comprises the following steps: firstly, selecting primary soybean oil with characteristic absorption wavelength of 360-860nm, and preparing a standard stock solution of copper chlorophyll by using the primary soybean oil as a diluent; secondly, preparing standard use solution of copper chlorophyll from blank olive oil without copper chlorophyll; thirdly, detecting the sample by adopting a spectrophotometer, and taking the absorbance of one wavelength or the average value of the absorbance of more than two wavelengths of 400-440nm or 620-660nm as the characteristic absorbance of the copper chlorophyll in the sample; fourthly, correcting the absorbance by using one or more wavelengths of 360-400nm, 440-540nm and 660-800 nm; and fifthly, quantifying by adopting an external standard method to obtain the content of the copper chlorophyll.
Further, the method for detecting the copper chlorophyll in the olive oil comprises the following steps: firstly, selecting soybean oil with no characteristic absorption wavelength of 653nm, and preparing a standard stock solution of copper chlorophyll by taking the soybean oil as a diluent; secondly, preparing standard use solution of copper chlorophyll from blank olive oil without copper chlorophyll; thirdly, detecting the sample by adopting a spectrophotometer, and taking the absorbance under the wavelength of 653nm as the characteristic absorbance of the sample; fourthly, correcting the absorbance by using one or two wavelengths of 671nm and 800 nm; and fifthly, quantifying by adopting an external standard method to obtain the content of the copper chlorophyll.
Wherein, the method for correcting the absorbance in the fourth step specifically comprises the following steps:
adopting 671nm wavelength to correct absorbance, wherein the method for correcting absorbance is 653nm absorbance/671 nm absorbance, the correction curve y =0.0737x +0.3441, R =0.9935, adding the same olive oil with the recovery rate of 85% -103%, the standard deviation of blank values S =0.0694, the detection limit LOD =3S/K =2.83mg/Kg, and K =0.0734 is the slope of a linear equation.
And B, correcting the absorbance by adopting a 671nm wavelength, wherein the wavelength correction absorbance method is 653nm absorbance-0.3 × 671nm absorbance, the correction curve y =0.034x +0.0139, R =0.9973, the addition recovery rate of the same olive oil is 88% -102%, the standard deviation of blank values S =0.0206, the detection limit LOD =3S/K =1.82mg/Kg, and K =0.034 is the slope of a linear equation.
And C, correcting the absorbance by adopting a 671nm wavelength, wherein the wavelength correction absorbance method is 653nm absorbance-0.3 absorbance-671 nm absorbance-0.7 absorbance 800 absorbance, the correction curve y =0.0337x-0.0032, R =0.9968, the addition recovery rate of the same olive oil is 85% -97%, the standard deviation of blank values is S =0.0241, the detection limit LOD =3S/K =2.15mg/Kg, and K =0.0337 is the slope of a linear equation.
Further, in the foregoing method for detecting copper chlorophyll in olive oil, the method for correcting absorbance for wavelength in the fourth step is 653nm absorbance-684 nm absorbance, the correction curve y =0.032x +0.0249, R =0.9969, the addition recovery rate for the same olive oil is 81% -97%, and the precision is 0.19-2.23%. Standard deviation of blank values S =0.0070, limit of detection LOD =3S/K =0.66mg/Kg, limit of quantitation LOQ =10S/K =2.19mg/Kg, K =0.032 is the slope of the linear equation. Three samples with large differences of the 671nm absorbance (one of the samples is minimum, one of the samples is in the middle and the other sample is maximum) are selected, and are respectively subjected to addition recovery at a detection limit, a quantification limit and a double quantification limit, wherein the addition recovery rate is 46-99%; the recovery rate of the added standard above the quantitative limit is 76-97%.
Further, in the method for detecting copper chlorophyll in olive oil, the method for correcting absorbance for wavelength in the fourth step is 653nm absorbance-683 nm absorbance/2-684 nm absorbance/2, the correction curve y =0.0319x +0.0173, R =0.9969, the addition recovery rate for the same olive oil is 81% -97%, and the precision is 0.19-2.16%. Standard deviation of blank values S =0.0061, limit of detection LOD =3S/K =0.57mg/Kg, limit of quantitation LOQ =10S/K =1.91mg/Kg, K =0.0319 is the slope of the linear equation. Selecting three samples with large differences of the 671nm absorbance (one of the samples is minimum, one of the samples is in the middle and the other sample is maximum), and respectively performing addition recovery at a detection limit, a quantification limit and a double quantification limit, wherein the addition recovery rate is 18-95%; the recovery rate of the added standard above the quantitative limit is 71-95%.
Further, in the foregoing method for detecting copper chlorophyll in olive oil, the method for correcting absorbance for wavelength in the fourth step is 653nm absorbance-684 nm absorbance/2-685 nm absorbance/2, the correction curve y =0.0321x +0.0312, R =0.9969, the addition recovery rate for the same olive oil is 81% -97%, and the precision is 0.20-2.19%. The standard deviation S =0.0104 for blank values, the limit of detection LOD =3S/K =0.97mg/Kg, the limit of quantitation LOQ =10S/K =3.24mg/Kg, K =0.0321 is the slope of the linear equation. Three samples with large differences of the 671nm absorbance (one of the samples is minimum, one of the samples is in the middle and the other sample is maximum) are selected, and added recovery is respectively carried out at the detection limit, the quantitative limit and the double quantitative limit, and the added standard recovery rate is 26-155 percent; the recovery rate of the added standard above the quantitative limit is 68-114%.
Further, in the method for detecting copper chlorophyll in olive oil, the method for correcting absorbance for wavelength in the fourth step is 653nm absorbance-683 nm absorbance/3-684 nm absorbance/3-685 nm absorbance/3, the correction curve y =0.032x +0.024, R =0.9969, the addition recovery rate for the same olive oil is 81% -97%, and the precision is 0.20-2.22%. Standard deviation of blank values S =0.0066, limit of detection LOD =3S/K =0.62mg/Kg, limit of quantitation LOQ =10S/K =2.06mg/Kg, K =0.032 is the linear equation slope. Selecting three samples with large differences of the 671nm absorbance (one of the samples is minimum, one of the samples is intermediate and one of the samples is maximum), and performing addition recovery at a detection limit, a quantification limit and a double quantification limit respectively, wherein the addition recovery rate is 48-96%; the recovery rate of the added standard above the quantitative limit is 77-96%.
Further, in the method for detecting copper chlorophyll in olive oil, one or more than two mixed solvents of petroleum ether, acetone and n-hexane are used as a standard stock solution and a standard use solution, a sample is dissolved in one or more than two mixed solvents of petroleum ether, acetone and n-hexane, and ultraviolet spectrophotometric analysis is performed; or saponifying the vegetable oil sample to be detected, extracting with one or more than two mixed solvents of petroleum ether, acetone and n-hexane, and performing ultraviolet spectrophotometry.
Further, in the method for detecting copper chlorophyll in olive oil, a vegetable oil sample to be detected is adsorbed by polyamide powder, and is analyzed by using an analysis solution, and then ultraviolet spectrophotometric analysis is performed.
Further, in the rapid qualitative screening and measuring method of the detection method of copper chlorophyll in olive oil, a full-wavelength collected olive oil sample spectrum is used, the 653nm absorbance and the 683nm absorbance are compared, if the 653nm absorbance is higher than the 683nm absorbance, or the 653nm absorbance is higher than the 683nm absorbance by 0.01-0.03AU, or is higher than 3 times of the standard deviation of the 653nm absorbance-683 absorbance, the sample is judged to be suspected to contain copper chlorophyll, and other methods are adopted for further confirmation and quantitative calculation.
Advantageous effects
After the technical scheme is adopted, the invention has the positive effects that:
the invention establishes a detection method for measuring the content of copper chlorophyll in olive oil by an ultraviolet-visible spectrophotometry, finds out the characteristic absorption light wavelength of copper chlorophyll through a larger soybean oil spectrogram, a copper chlorophyll standard stock solution spectrogram and a blank olive oil spectrogram, the characteristic wavelength is 653nm, carries out comparison tests of 7 different correction methods, comprehensively considers the factors of detection limit, quantitative limit, correction curve, standard deviation, standard recovery rate and the like, determines the optimal correction method, eliminates matrix interference and ensures the accuracy of the detection result.
The invention adopts a common spectrophotometer, and the pretreatment is simple: only the sample is poured into the cuvette, and complex pretreatment equipment is not needed, so that the method is convenient to popularize; the detection is quick: judging the result immediately after detection; sensitivity: the lowest detection limit can reach 0.57mg/Kg, and the quantification limit is 1.91 mg/Kg; the specificity is high: because various absorbances are adopted for correction in the detection process, the result is accurate; the price is cheap: the cost is greatly lower than that of a chromatographic instrument and a mass spectrometer, the method is easy to popularize in plant oil manufacturers, plant oil wholesale retail enterprises and detection mechanisms, and the method has wide application prospect in the field of food safety detection. The determination of the content of copper chlorophyll in other common foods can be completed by adopting a method similar to the method.
The invention is novel in that the invention discloses a visible light spectrophotometric detection method of copper chlorophyll in olive oil, and has the advantages of intuition, rapidness, convenience, maturity, low price and the like.
Drawings
FIG. 1 is a characteristic absorption spectrum of a first-grade soybean oil.
FIG. 2 is a spectrum of standard stock solution of copper chlorophyll.
Figure 3 blank olive oil spectrum.
FIG. 4 is a comparison of soybean oil spectrum, chlorophyll copper standard stock solution spectrum, and blank olive oil spectrum.
FIG. 5 illustrates a standard curve.
FIG. 6 shows a second calibration curve for the example.
Fig. 7 example three standard curves.
Figure 8 example four standard curves.
Fig. 9 example five standard curves.
Fig. 10 example six standard curves.
Fig. 11 illustrates a seven-standard curve for the example.
Detailed Description
The present invention will be described in further detail below with reference to preferred embodiments.
1. Preparation of standard solution of copper chlorophyll
1.1 determination of concentration of copper chlorophyll Standard solution
Since no standard substance of copper chlorophyll is commercially available, only food additives are purchased as standard substances, but the content is marked as a color scale, and the color value measurement method is a method for measuring the color value of the concentration of a pigment in a colorant by measuring absorbance [9], and not mass concentration, and the method for measuring the bound copper is adopted herein to calculate the content of copper chlorophyll.
1.1.1 total copper content 0.1g of sample is accurately weighed, accurately to 0.0002g, placed in a silicon dish, burned to be carbon-free at the temperature of no more than 500 ℃, moistened by 1-2 drops of sulfuric acid, and burned again. The ash content was boiled and dissolved 3 times (5 mL each) with a 10% hydrochloric acid solution, filtered in a 100mL volumetric flask, and after cooling, the volume was fixed to the scale with water, which was a sample solution. The procedure was as defined in GB/T5009.13 except for the sample treatment.
1.1.2 sample treatment of free copper content: weighing about 0.1g of sample, accurately obtaining 0.0002g of sample, dissolving the sample in an erlenmeyer flask by using 20mL of anhydrous ether, adding 100mL of water, plugging, shaking for 1min, transferring the sample into a 125mL separating funnel, standing for 30min, filtering the lower-layer aqueous solution in the separating funnel by using double-layer qualitative filter paper, and if the filtrate is colored, filtering by using the double-layer qualitative filter paper again to obtain the filtrate, namely the sample solution. The procedure was as defined in GB/T5009.13 except for the sample treatment.
1.1.3 copper content of copper chlorophyll = total copper content-free copper content
1.1.4 content of copper chlorophyll = copper content of copper chlorophyll/copper molecular weight copper chlorophyll molecular weight
The content of copper chlorophyll in the food additive with color level of 10 is detected to be 902.6mg/kg.
1.1.5 the standard stock solution of copper chlorophyll is prepared from soybean oil (shown in a spectrogram in figure 1) with a characteristic absorption wavelength of 360-860nm as a diluent, and the standard stock solution is prepared by a precision electronic balance weighing method, wherein the concentration of the standard stock solution is 65.7mg/kg, and the spectrogram is shown in figure 2.
1.1.6 Standard solution of copper chlorophyll
Preparing standard use solution of copper chlorophyll from blank olive oil without copper chlorophyll, and preparing the standard use solution by using a precision electronic balance weighing method, wherein the concentrations are respectively 0, 0.7, 2.0, 3.3, 4.3 and 6.5mg/kg.
2. Sample assay
2.1 instruments and reagents
Agilent UV-visible spectrophotometer 8453, diode array detector, 1cm quartz cuvette, XS205Du electronic balance (Mettler-Toledo, Switzerland).
Edible vegetable oil samples were collected from the market or manufacturer in 18 pieces, including 13 soybean oil (Soybeanoil) and 5 palm oil (Palmoil).
Food additive copper chlorophyll (color value E10, Hay DaBiotech Co., Ltd., Fushan city).
2.2 detection method the standard use solution and the sample were poured into a 1cm cuvette and scanned with 190-1100nm full band.
3. The quantitative calculation adopts an external standard method to calculate the content of the copper chlorophyll.
As can be seen from the soybean oil spectrogram (figure 1), the copper chlorophyll standard stock solution spectrogram (figure 2), the blank olive oil spectrogram (figure 3) and the comparison among the three spectrograms (figure 4), the copper chlorophyll has a characteristic absorption spectrum which is respectively at 400-440nm or 620-660nm and can be used for qualitative and quantitative detection; in addition, no characteristic absorption at 360-400nm, 440-540nm and 660-800nm can be used as the calibration spectrum. And (3) respectively adopting the absorbances of wavelengths of 671nm, 800nm, 683nm, 684nm, 685nm and the like to correct the absorbances of the characteristic absorption wavelength 653nm, and respectively inspecting the aspects of a standard curve, an addition recovery rate, a standard deviation of a blank value, a detection limit and the like to determine a correction wavelength and a correction method.
The invention is explained in further detail below with reference to nine examples. A standard curve is made by adding copper chlorophyll with different concentrations into a special virgin olive oil, and an addition recovery experiment and an accuracy test are made by adding three-level six-parallel marks of the special virgin olive oil. And taking another 11 extra-grade virgin olive oils to repeatedly measure blank values for 22 times, measuring the relative standard deviation of the blank values, and calculating detection limit and quantitative limit. Three samples with different representatives at 671nm (one of the samples has the highest absorbance, one of the samples has the lowest absorbance, and one of the samples has the middle absorbance) are taken and respectively taken as an addition recovery experiment at 1-time detection limit, a quantification limit and a 2-time quantification limit, and the selectivity of the method is evaluated.
Example one
The absorbance was corrected with the 671nm wavelength by 653nm absorbance/671 nm absorbance, the correction curve y =0.0737x +0.3441, R =0.9935, the addition recovery of the same olive oil was 85% -103%, the standard deviation of blank values S =0.0694, the detection limit LOD =3S/K =2.83mg/Kg, K =0.0734 is the slope of the linear equation. The detailed data are shown in tables 1-3.
Table 1 example a standard curve data
| Name (R) | Abs<653nm> | Abs<671nm> | Concentration (mg/kg) | Abs<653nm>/Abs<671nm> |
| st0-1 | 0.13582 | 0.39922 | 0 | 0.3402 |
| st0-2 | 0.13482 | 0.39822 | 0 | 0.3386 |
| st1-1 | 0.15311 | 0.3981 | 0.7142 | 0.3846 |
| st1-2 | 0.15656 | 0.40219 | 0.7142 | 0.3893 |
| st2-1 | 0.2014 | 0.41126 | 1.9911 | 0.4897 |
| st2-2 | 0.20419 | 0.41464 | 1.9911 | 0.4925 |
| st3-1 | 0.25263 | 0.4218 | 3.3053 | 0.5989 |
| st3-2 | 0.25802 | 0.42765 | 3.3053 | 0.6033 |
| st4-1 | 0.29759 | 0.43772 | 4.2769 | 0.6799 |
| st4-2 | 0.29765 | 0.43862 | 4.2769 | 0.6786 |
| st5-1 | 0.3692 | 0.45682 | 6.5410 | 0.8082 |
| st5-2 | 0.37043 | 0.45869 | 6.5410 | 0.8076 |
Table 2 example one addition recovery experimental data
Table 3 example a blank standard deviation experimental data
Example two
The 671nm wavelength is adopted to correct the absorbance, the wavelength correction absorbance method is 653nm absorbance-0.3 × 671nm absorbance, the correction curve y =0.034x +0.0139, R =0.9973, the addition recovery rate to the same olive oil is 88% -102%, the standard deviation of blank values S =0.0206, the detection limit LOD =3S/K =1.82mg/Kg, and K =0.034 is the slope of a linear equation.
Table 4 example two standard curve data
| Name (R) | Abs<653nm> | Abs<671nm> | Concentration (mg/kg) | Wavelength correction value |
| st0-1 | 0.13582 | 0.39922 | 0 | 0.0161 |
| st0-2 | 0.13482 | 0.39822 | 0 | 0.0154 |
| st1-1 | 0.15311 | 0.3981 | 0.7142 | 0.0337 |
| st1-2 | 0.15656 | 0.40219 | 0.7142 | 0.0359 |
| st2-1 | 0.2014 | 0.41126 | 1.9911 | 0.0780 |
| st2-2 | 0.20419 | 0.41464 | 1.9911 | 0.0798 |
| st3-1 | 0.25263 | 0.4218 | 3.3053 | 0.1261 |
| st3-2 | 0.25802 | 0.42765 | 3.3053 | 0.1297 |
| st4-1 | 0.29759 | 0.43772 | 4.2769 | 0.1663 |
| st4-2 | 0.29765 | 0.43862 | 4.2769 | 0.1661 |
| st5-1 | 0.3692 | 0.45682 | 6.5410 | 0.2322 |
| st5-2 | 0.37043 | 0.45869 | 6.5410 | 0.2328 |
Table 5 example two addition recovery experimental data
Table 6 experimental data for standard deviation of blank values of example two
EXAMPLE III
The absorbance is corrected by adopting wavelengths of 671nm and 800nm, wherein the absorbance correction method is 653nm absorbance-0.3 × 671nm absorbance-0.7 × 800 absorbance, the correction curve y =0.0337x-0.0032, R =0.9968, the addition recovery rate of the same olive oil is 85% -97%, the standard deviation of blank values is S =0.0241, the detection limit LOD =3S/K =2.15mg/Kg, and K =0.0337 is the slope of a linear equation.
TABLE 7 data of the three standard curves of the examples
Table 8 example iii addition recovery experimental data
TABLE 9 data of the standard deviation test of the three blank values of the examples
Example four
The 684nm wavelength is adopted to correct the absorbance, the method for correcting the absorbance is 653nm absorbance-684 nm absorbance, the correction curve y =0.032x +0.0249, R =0.9969, the addition recovery rate of the same olive oil is 81% -97%, and the precision is 0.19-2.23%. Standard deviation of blank values S =0.0070, limit of detection LOD =3S/K =0.66mg/Kg, limit of quantitation LOQ =10S/K =2.19mg/Kg, K =0.032 is the slope of the linear equation. Three samples with large differences of the 671nm absorbance (one of the samples is minimum, one of the samples is in the middle and the other sample is maximum) are selected, and are respectively subjected to addition recovery at a detection limit, a quantification limit and a double quantification limit, wherein the addition recovery rate is 46-99%; the recovery rate of the added standard above the quantitative limit is 76-97%.
TABLE 10 data of the four standard curves of the examples
Table 11 example four addition recovery experimental data
TABLE 12 data of standard deviation of four blank values for the examples
Table 13 example four different sample addition recovery experimental data
EXAMPLE five
The 683nm and 684nm wavelengths are adopted to correct the absorbance, the method for correcting the absorbance is 653nm absorbance-683 nm absorbance/2-684 nm absorbance/2, the correction curve y =0.0319x +0.0173, R =0.9969, the addition recovery rate of the same olive oil is 81% -97%, and the precision is 0.19-2.16%. Standard deviation of blank values S =0.0061, limit of detection LOD =3S/K =0.57mg/Kg, limit of quantitation LOQ =10S/K =1.91mg/Kg, K =0.0319 is the slope of the linear equation. Selecting three samples with large differences of the 671nm absorbance (one of the samples is minimum, one of the samples is in the middle and the other sample is maximum), and respectively performing addition recovery at a detection limit, a quantification limit and a double quantification limit, wherein the addition recovery rate is 18-95%; the recovery rate of the added standard above the quantitative limit is 71-95%.
Table 14 example five standard curve data
Table 15 example five addition recovery experimental data
TABLE 16 Standard deviation experimental data for blank values of the examples
Table 17 example five different sample addition recovery experimental data
EXAMPLE six
The wavelengths of 684nm and 685nm are adopted for correcting the absorbance, the method for correcting the absorbance is 653nm absorbance-684 nm absorbance/2-685 nm absorbance/2, the correction curve y =0.0321x +0.0312, R =0.9969, the adding recovery rate of the same olive oil is 81% -97%, and the precision is 0.20-2.19%. The standard deviation S =0.0104 for blank values, the limit of detection LOD =3S/K =0.97mg/Kg, the limit of quantitation LOQ =10S/K =3.24mg/Kg, K =0.0321 is the slope of the linear equation. Three samples with large differences of the 671nm absorbance (one of the samples is minimum, one of the samples is in the middle and the other sample is maximum) are selected, and added recovery is respectively carried out at the detection limit, the quantitative limit and the double quantitative limit, and the added standard recovery rate is 26-155 percent; the recovery rate of the added standard above the quantitative limit is 68-114%.
Table 18 example six standard curve data
Table 19 example six additive recovery experimental data
TABLE 20 example six blank standard deviation experimental data
Table 21 example six different sample addition recovery experimental data
EXAMPLE seven
The 683nm, 684nm and 685nm wavelengths are used for correcting the absorbance, the method for correcting the absorbance is 653nm absorbance-683 nm absorbance/3-684 nm absorbance/3-685 nm absorbance/3, the correction curve y =0.032x +0.024, R =0.9969, the addition recovery rate of the same olive oil is 81% -97%, and the precision is 0.20-2.22%. Standard deviation of blank values S =0.0066, limit of detection LOD =3S/K =0.62mg/Kg, limit of quantitation LOQ =10S/K =2.06mg/Kg, K =0.032 is the linear equation slope. Selecting three samples with large differences of the 671nm absorbance (one of the samples is minimum, one of the samples is intermediate and one of the samples is maximum), and performing addition recovery at a detection limit, a quantification limit and a double quantification limit respectively, wherein the addition recovery rate is 48-96%; the recovery rate of the added standard above the quantitative limit is 77-96%.
TABLE 22 data of the seven standard curves of the examples
Table 23 example seven addition recovery experimental data
TABLE 24 Standard deviation experimental data for seven blank values of the examples
Table 25 example seven different sample addition recovery experimental data
In example eight, in the method for detecting copper chlorophyll in olive oil, a vegetable oil sample to be detected is adsorbed by polyamide powder, and is analyzed by an analysis solution, and then is subjected to ultraviolet spectrophotometric analysis. Dissolving 5.0g of sample in 15mL of one or more than two of petroleum ether, acetone and normal hexane mixed solvent, adding 3.0g of polyamide powder, wherein the mesh number of the polyamide powder is 100-200 meshes, and the adding amount of the polyamide powder needs to ensure that the chlorophyll copper in the extracting solution can be fully adsorbed. And (3) uniformly mixing the extracting solution added with the polyamide powder for 2min in a vortex manner, so that the polyamide powder can fully adsorb the copper chlorophyll in the extracting solution, wherein an instrument adopted by the uniform mixing in the vortex manner is a vortex mixer, and the uniform mixing in the vortex manner is used for enabling the polyamide powder to better fully adsorb the copper chlorophyll in the extracting solution. Then, the extract and the polyamide powder adsorbed with copper chlorophyll were transferred to a G4 sand core funnel and filtered with 20mL of anhydrous ethanol to obtain the polyamide powder adsorbed with copper chlorophyll. And thirdly, dividing 45mL of desorption solution into four parts, wherein the amount of each part of desorption solution is required to ensure that the copper chlorophyll adsorbed on the polyamide powder can be fully desorbed. The four desorption solutions respectively carry out one-time desorption on the polyamide powder adsorbed with the copper chlorophyll, and the four desorption solutions are combined to carry out four desorption, so that the purpose of carrying out four desorption is as follows: ensuring sufficient desorption of the copper chlorophyll adsorbed on the polyamide powder. And transferring the combined copper chlorophyllin solution to a volumetric flask with a desorption solution to reach a constant volume of 50mL for testing. The desorption solution is formed by mixing 0.1mol/L sodium hydroxide aqueous solution and anhydrous methanol, and the volume ratio of the 0.1mol/L sodium hydroxide aqueous solution to the anhydrous methanol is 1: 10.
The nine quick qualitative screening and determining method comprises the steps of collecting a spectrum of an olive oil sample by using full wavelength, judging that the sample is suspected to contain copper chlorophyll if the 653nm absorbance is higher than 683nm absorbance, or the 653nm absorbance is higher than 683nm absorbance by 0.01-0.03AU, or is higher than 653nm absorbance-3 times of standard deviation of 683 absorbance, and further carrying out further confirmation and quantitative calculation by other modes. The standard deviation of 653-683 absorbance was calculated according to the same method as in example four. As can be seen from the blank olive oil spectrogram (FIG. 3) and Table 24, 653nm and 683nm are respectively located at two sides of the main absorption peak of the blank olive oil, and the 683nm absorbance is slightly higher than the 653nm absorbance, and if the 653nm absorbance is higher than the 683nm absorbance, the illegal addition of copper chlorophyll is possible.
For examples 1 to 7, the detection limit, the quantitation limit, the calibration curve, the standard deviation, the standard recovery rate and other factors are comprehensively considered, and the optimal method is to select the calibration method 7 as the detection method of the copper chlorophyll in the olive oil, namely, the characteristic wavelength of 653nm absorbance-683 nm absorbance/3-684 nm absorbance/3-685 nm absorbance/3, the calibration curve y =0.032x +0.024, and R =0.9969, and the recovery rate of the same olive oil is 81% to 97%, and the precision is 0.20% to 2.22%. Standard deviation of blank values S =0.0066, limit of detection LOD =3S/K =0.62mg/Kg, limit of quantitation LOQ =10S/K =2.06mg/Kg, K =0.032 is the linear equation slope. Selecting three samples with large differences of the 671nm absorbance, and respectively performing addition recovery at a detection limit, a quantification limit and a double quantification limit, wherein the detection limit standard addition recovery rate is 48-90%; the recovery rate of the quantitative limit adding standard is 77-93%, and the recovery rate of the double quantitative limit adding standard is 89-96%.
Claims (9)
1. A method for detecting copper chlorophyll in olive oil is characterized by comprising the following steps: firstly, selecting primary soybean oil with characteristic absorption wavelength of 360-860nm, and preparing a standard stock solution of copper chlorophyll by using the primary soybean oil as a diluent; calculating the content of copper chlorophyll by adopting a method for measuring bound copper, determining the concentration of a standard solution of copper chlorophyll, and preparing a standard use solution of copper chlorophyll from blank olive oil which does not contain copper chlorophyll; thirdly, detecting the sample by adopting a spectrophotometer, and taking the absorbance of one wavelength or the average value of the absorbance of more than two wavelengths of 400-440nm or 620-660nm as the characteristic absorbance of the copper chlorophyll in the sample; fourthly, correcting the absorbance by using one or more wavelengths of 360-400nm, 440-540nm and 660-800 nm; fifthly, quantifying by adopting an external standard method to obtain the content of the copper chlorophyll;
the method for calculating the content of the copper chlorophyll by adopting the method for measuring the bound copper comprises the following specific steps:
1) accurately weighing 0.1g of sample with the total copper content, accurately measuring the sample to 0.0002g, placing the sample in a silicon dish, burning the sample until no carbon exists at the temperature of no more than 500 ℃, wetting the sample by using 1-2 drops of sulfuric acid, burning again, dividing the sample into 5mL portions by using 10% hydrochloric acid solution by mass fraction for 3 times, boiling the solution to dissolve ash, filtering the solution in a 100mL volumetric flask, cooling the solution, and then using water to fix the volume to a scale, wherein the sample is sample liquid, except sample treatment, other steps are measured according to the method specified in GB/T5009.13,
2) treating a free copper content sample: weighing about 0.1g of sample, accurately measuring to 0.0002g, dissolving in 20mL of anhydrous ether in an erlenmeyer flask, adding 100mL of water, plugging, shaking for 1min, transferring into a 125mL separating funnel, standing for 30min, filtering the lower layer aqueous solution in the separating funnel by using double-layer qualitative filter paper, if the filtrate has color, filtering by using double-layer qualitative filter paper again to obtain the filtrate, namely the sample solution, measuring the other steps according to the method specified in GB/T5009.13 except sample treatment,
3) copper content of copper chlorophyll = total copper content-free copper content,
4) copper chlorophyll content = copper chlorophyll content/copper molecular weight copper chlorophyll molecular weight.
2. The method for detecting copper chlorophyll in olive oil according to claim 1, wherein the first-stage soybean oil with a characteristic absorption wavelength of 360-860nm is selected as soybean oil with a characteristic absorption wavelength of 653nm, and the first-stage soybean oil is used as a diluent to prepare a standard stock solution of copper chlorophyll; and step three, taking the average value of the absorbance at one wavelength or more than two wavelengths of 400-440nm or 620-660nm as the characteristic absorbance of the copper chlorophyll in the sample, and taking the absorbance at 653nm as the characteristic absorbance of the sample.
3. The method for detecting copper chlorophyll in olive oil according to claim 2, wherein the method for correcting absorbance in the fourth step is specifically as follows: the absorbance was corrected with the 671nm wavelength by 653nm absorbance/671 nm absorbance, the correction curve y =0.0737x +0.3441, R =0.9935, the addition recovery of the same olive oil was 85% -103%, the standard deviation of blank values S =0.0694, the detection limit LOD =3S/K =2.83mg/Kg, K =0.0734 is the slope of the linear equation.
4. The method for detecting copper chlorophyll in olive oil according to claim 2, wherein the method for correcting absorbance in the fourth step is specifically as follows: the 671nm wavelength is adopted to correct the absorbance, the wavelength correction absorbance method is 653nm absorbance-0.3 × 671nm absorbance, the correction curve y =0.034x +0.0139, R =0.9973, the addition recovery rate to the same olive oil is 88% -102%, the standard deviation of blank values S =0.0206, the detection limit LOD =3S/K =1.82mg/Kg, and K =0.034 is the slope of a linear equation.
5. The method for detecting copper chlorophyll in olive oil according to claim 2, wherein the method for correcting absorbance in the fourth step is specifically as follows: the 671nm wavelength is adopted to correct the absorbance, the wavelength correction absorbance method is 653nm absorbance-0.3 × 671nm absorbance-0.7 × 800 absorbance, the correction curve y =0.0337x-0.0032, R =0.9968, the addition recovery rate of the same olive oil is 85% -97%, the standard deviation S =0.0241 of blank values, the detection limit LOD =3S/K =2.15mg/Kg, and K =0.0337 is the slope of a linear equation.
6. The method according to claim 2, wherein the wavelength-corrected absorbance method in step four is 653nm absorbance-684 nm absorbance, the correction curve y =0.032x +0.0249, R =0.9969, the addition recovery rate for the same olive oil is 81% -97%, the precision is 0.19-2.23%, the standard deviation of blank values S =0.0070, the detection limit LOD =3S/K =0.66mg/Kg, the quantification limit LOQ =10S/K =2.19mg/Kg, K =0.032 is the slope of a linear equation, and the addition recovery rate is 46% -99%; the recovery rate of the added standard above the quantitative limit is 76-97%.
7. The method for detecting copper chlorophyll in olive oil according to claim 2, wherein the wavelength-corrected absorbance method in step four is 653nm absorbance-683 nm absorbance/2-684 nm absorbance/2, the correction curve y =0.0319x +0.0173, R =0.9969, the addition recovery rate for the same olive oil is 81% -97%, the precision is 0.19-2.16%, the standard deviation of blank values S =0.0061, the detection limit LOD =3S/K =0.57mg/Kg, the quantification limit LOQ =10S/K =1.91mg/Kg, and K =0.0319 is the slope of a linear equation; the recovery rate of the added standard is 18-95 percent; the recovery rate of the added standard above the quantitative limit is 71-95%.
8. The method for detecting copper chlorophyll in olive oil according to claim 2, wherein the wavelength-corrected absorbance method of step four is 653nm absorbance-684 nm absorbance/2-685 nm absorbance/2, the correction curve y =0.0321x +0.0312, R =0.9969, the addition recovery rate for the same olive oil is 81% -97%, and the precision is 0.20-2.19%; standard deviation S =0.0104 for blank values, limit of detection LOD =3S/K =0.97mg/Kg, limit of quantitation LOQ =10S/K =3.24mg/Kg, K =0.0321 is the slope of the linear equation; the recovery rate of the added standard is 26-155 percent; the recovery rate of the added standard above the quantitative limit is 68-114%.
9. The method for detecting copper chlorophyll in olive oil according to claim 2, wherein the wavelength-corrected absorbance method of step four is 653nm absorbance-683 nm absorbance/3-684 nm absorbance/3-685 nm absorbance/3, the correction curve y =0.032x +0.024, R =0.9969, the addition recovery rate of the same olive oil is 81% -97%, and the precision is 0.20-2.22%; standard deviation of blank values S =0.0066, limit of detection LOD =3S/K =0.62mg/Kg, limit of quantitation LOQ =10S/K =2.06mg/Kg, K =0.032 is the slope of the linear equation; the recovery rate of the added standard is 48-96%; the recovery rate of the added standard above the quantitative limit is 77-96%.
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