CN113203614B - Method for determining calcium carbonate in reconstituted tobacco by applying enzymatic reaction - Google Patents
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Abstract
The invention discloses a method for determining calcium carbonate in reconstituted tobacco by applying enzymatic reaction, which comprises the following steps: s1, regeneratingGrinding tobacco leaves into tobacco powder, and drying to constant weight for later use; s2, weighing the tobacco powder prepared in the step S1, putting the tobacco powder into a reactor, adding deionized water, adding carbonic anhydrase, and introducing CO2Carrying out reaction, sealing the reactor after the reaction is finished, placing the reactor in an ice bath for oscillation, and then filtering to obtain a solution to be detected; s3, diluting the solution to be measured with ice water, and measuring Ca by using atomic absorption spectrophotometer2+Then the absorbance is brought into a standard working curve to obtain Ca in the solution to be detected2+The concentration of (3) C; s4, calculating Ca in the blank sample2+Concentration C0(ii) a And S5, calculating the content of calcium carbonate in the reconstituted tobacco according to a formula. Introducing CO into the water extract of the reconstituted tobacco2The calcium carbonate can be changed into calcium bicarbonate as a target object to be extracted, meanwhile, the dissolution of other calcium salts in the reconstituted tobacco can be avoided, the extraction accuracy is high, and the reliability is strong.
Description
Technical Field
The invention relates to the technical field of analysis of calcium carbonate content in reconstituted tobacco, in particular to a method for determining calcium carbonate in reconstituted tobacco by applying enzymatic reaction.
Background
Reconstituted Tobacco (Reconstituted tobaccos) is a sheet-shaped or filiform regenerated product prepared from Tobacco materials such as Tobacco powder, Tobacco stems, Tobacco shreds and the like serving as raw materials and used as a cigarette filler. The paper-making reconstituted tobacco is added into the cigarettes, so that the tobacco resources can be fully utilized, the production cost is reduced, the tar content of the cigarettes can be effectively reduced, and the method is an effective way for improving the safety of the cigarettes. The production process of the paper-making reconstituted tobacco mainly comprises main processes of extraction, concentration, pulping, papermaking, coating, drying and the like, and a certain amount of calcium carbonate is usually added in the production process to improve the ash content so as to improve the physical properties, such as liquid absorption performance, appearance performance and the like. Therefore, the content of calcium carbonate in the reconstituted tobacco is accurately and quickly determined, and the product appearance, the smoke condition and the like are conveniently evaluated. The main methods for measuring calcium carbonate at present are complexation titration method, potentiometric titration method, continuous flow method and ion chromatography. The measuring process of the complexometric titration method and the potentiometric titration method is complicated, and human errors are easily introduced; the ion chromatography and the continuous flow method are both used for measuring the mass fraction of acid-soluble total calcium, and calcium element in the reconstituted tobacco coexists in various forms (water-soluble inorganic calcium, organic calcium, calcium phosphate, calcium oxalate and calcium carbonate), and calcium in different forms sometimes dissolves out together in an acid solution, so that the two methods cannot accurately reflect the mass fraction of calcium carbonate in a sample.
Chinese patent (CN 102830174A) discloses a method for rapidly identifying the content of calcium carbonate in paper-making reconstituted tobacco, which is characterized in that the reconstituted tobacco is fixed in a sample tank filled with acid solution, an ultrasonic transmitter is arranged at one side of the sample tank, an ultrasonic receiver is arranged at the other side of the sample tank, ultrasonic waves transmitted by the ultrasonic transmitter penetrate through the reconstituted tobacco and are received by the ultrasonic receiver, and the content of calcium carbonate in the reconstituted tobacco can be identified by detecting a signal attenuation value received by the ultrasonic receiver. The method has problems in that the specific content of calcium carbonate cannot be accurately obtained, and the influence of carbonate contained in tobacco itself is not considered.
Chinese patent No. CN102901791A discloses a method for measuring the content of calcium carbonate in reconstituted tobacco, which comprises the steps of firstly extracting the reconstituted tobacco by using excessive acetic acid solution, measuring the total calcium ion content by using a modern analysis method, then analyzing the content of free calcium ions in the reconstituted tobacco extracted by deionized water under the same condition, and calculating the content of calcium carbonate in the reconstituted tobacco by using the difference of the two contents. The method has the problems that the reconstituted tobacco contains a large amount of calcium pectate which is insoluble in water and soluble in acetic acid, and the calcium in the calcium pectate is calculated as calcium carbonate according to the measurement result, so that the measurement result is higher.
Disclosure of Invention
In order to solve the problems, the invention provides a method for determining calcium carbonate in reconstituted tobacco by applying enzymatic reaction, and the method is used for applying the enzymatic reaction to the reconstituted tobaccoAdding carbonic anhydrase into water extractive solution of leaf, and introducing CO2Oscillating in ice bath to react with calcium carbonate to generate soluble calcium bicarbonate, filtering and diluting, and measuring Ca by atomic absorption method2+The absorbance of (A) is substituted into a standard working curve to obtain Ca2+Then Ca is calculated by a formula2+The content of (a).
In order to achieve the purpose, the technical scheme provided by the invention is as follows: a method for determining calcium carbonate in reconstituted tobacco by applying enzymatic reaction is characterized by comprising the following steps:
s1, grinding the reconstituted tobacco into tobacco powder, and drying to constant weight for later use;
s2, weighing the tobacco powder prepared in the step S1, putting the tobacco powder into a reactor, adding deionized water, adding carbonic anhydrase, and introducing CO2Carrying out reaction, sealing the reactor after the reaction is finished, placing the reactor in an ice bath for oscillation, and then filtering to obtain a solution to be detected;
s3, diluting the solution to be detected obtained in the step S2 with ice water, and measuring Ca by using an atomic absorption spectrophotometer2+Then the absorbance is brought into a standard working curve to obtain Ca in the solution to be detected2+The concentration of (3) C;
the manufacturing method of the standard working curve comprises the following steps: preparing Ca with different concentrations2+Standard solution, measuring Ca with different concentrations by atomic absorption spectrophotometer2+Then Ca2+The concentration is an abscissa, and a standard working curve is drawn by taking the corresponding absorbance as an ordinate;
s4, Ca in blank sample2+Concentration C0The calculating method of (2): weighing the tobacco powder prepared in the step S1, placing the tobacco powder into a reactor, adding deionized water, adding carbonic anhydrase for reaction, sealing the reactor after the reaction is finished, placing the reactor into an ice bath for oscillation, then filtering to obtain a blank liquid to be detected, diluting the blank liquid to be detected with ice water, and measuring Ca by using an atomic absorption spectrophotometer2+Then bringing the absorbance into a standard working curve to obtain Ca in the blank solution to be tested2+Concentration C of0；
S5, calculating the content of calcium carbonate in the reconstituted tobacco according to the following formula:
in the above formula:
x represents the content of calcium carbonate in the reconstituted tobacco, and the unit is microgram per gram (mu g/g);
c-concentration of target in micrograms per milliliter (. mu.g/mL) from the standard working curve;
C0-target concentration in micrograms per milliliter (μ g/mL) in a blank sample from a standard working curve;
v-volume of extract in milliliters (mL);
f is the dilution factor;
m-sample mass in grams (g);
100——CaCO3the formula (2);
40——Ca2+the formula (2).
Further, the mass ratio of the tobacco powder, the deionized water and the carbonic anhydrase in the steps S2 and S4 is (2000- & 4000): (500000-1000000): 1.
Further, in the step S2, CO is introduced2The time is 20-60 min, and CO is introduced2The flow rate of (A) is 0.5 to 2.0 mL/min.
Further, the ice bath temperature in the steps S2 and S4 is 0-4 ℃.
Further, the oscillation time in the steps S2 and S4 is 20-60 min.
Further, the solution to be tested in the step S3 and the hollow white solution to be tested in the step S4 are both diluted by 90-110 times with ice water.
The invention has the beneficial effects that:
1. the invention provides a method for determining calcium carbonate in reconstituted tobacco by applying enzymatic reaction, which is characterized in that CO is introduced into a water extracting solution of the reconstituted tobacco2Not only can change the calcium carbonate of the target object into calcium bicarbonate to be extracted, but also can avoid the reconstituted tobaccoThe dissolution of other calcium salts (calcium phosphate, calcium oxalate and calcium sulfate) in the leaves has high extraction accuracy and strong reliability. The method has the characteristics of small interference, high sensitivity, no reagent pollution and the like, and can be applied to the determination of the content of calcium carbonate in various calcium salt mixed systems.
2. The method for determining calcium carbonate in reconstituted tobacco by applying enzymatic reaction provided by the invention can catalyze CO by adding carbonic anhydrase2Hydration reaction, which makes it proceed quickly under physiological pH value condition (pH value ≈ 7), accelerates bicarbonate radical conversion rate, and thus improves detection rate.
3. The method for determining calcium carbonate in reconstituted tobacco by applying enzymatic reaction provided by the invention adopts ice bath oscillation to ensure that calcium carbonate and CO can be mixed2The reaction system maintains a lower temperature, not only can increase CO2The solubility in water can also ensure the stability of calcium bicarbonate.
Drawings
FIG. 1 is a flow chart of a method for determining calcium carbonate in reconstituted tobacco by applying enzymatic reaction in the embodiment of the invention.
Detailed Description
In order to more clearly illustrate the embodiments of the present invention and the technical solutions in the prior art, the following will describe the specific embodiments of the present invention with reference to the accompanying drawings.
It is obvious that the drawings in the following description are only some examples of the invention, and it is obvious to a person skilled in the art that other drawings and other embodiments can be obtained from these drawings without inventive effort, and the invention is not limited to this example.
In the embodiment of the invention: the operating conditions of the apparatus are as follows: wavelength: 422.7 nm; lamp current: 10 mA; acetylene flow rate: 2.0L/min; air flow rate: 10L/min.
The specific embodiment of the invention is as follows:
example 1
And (3) preparing a standard curve:
the prepared concentrations were 0.1mg/L, 1.0 mg/L, 2.0 mg/L, 3.0 mg/L, 4.0 mg/LSix gradients of calcium ion standard solution of/L and 5.0mg/L, and measuring Ca of the six standard solutions by using an atomic absorption spectrophotometer2+Then Ca2+And (3) drawing a standard working curve by taking the concentration as an abscissa and the corresponding absorbance as an ordinate, and calculating a linear equation. Obtaining a linear equation Y =0.0433X +0.0003, wherein Y is absorbance and X is Ca2+And the correlation coefficient of the concentration is 0.9997, the correlation coefficient is close to 1, and the explanation error is small, which shows that the working curve has a good linear fitting relation in the range of 0.1-5.0 mg/L. The Ca in the solution to be detected can be directly obtained by introducing the linear equation into the absorbance in the later period2+And (4) concentration.
Detection limit and quantification limit:
detection limit: the assay detects the lowest concentration or amount of the component to be detected under the specified experimental conditions.
And (4) quantitative limit: the assay allows the lowest concentration or amount of the component to be measured in the sample to be quantitatively determined.
Taking 0.1mg/L calcium ion standard solution as an analysis object, measuring eight times by adopting an atomic absorption spectrophotometer, and substituting the eight measurement results into a standard curve to obtain corresponding Ca2+The standard deviation of the measured values was calculated, and the results of conversion into 3-fold standard deviation were used as the detection Limit (LOD) of the method, and the results of conversion into 10-fold standard deviation were used as the quantification Limit (LOQ) of the method, and the results are shown in Table 1.
TABLE 1 detection and quantitation limits
As can be seen from Table 1, a standard deviation of 0.00529, the smaller the standard deviation, the smaller the difference between the measured value and the average value, indicating the higher the accuracy of the calculation of the calibration curve. The limit of detection (LOD) was 0.01586 mg/L, which corresponds to 0.9911mg/g, indicating that the minimum detectable concentration for this method was 0.9911 mg/g. The limit of quantitation (LOQ) is 0.05286mg/L, which corresponds to 3.3035mg/g, indicating that the lowest concentration that can be detected by this method is 3.3035 mg/g. Can meet the detection requirement of calcium carbonate in the reconstituted tobacco.
Recovery and relative standard deviation of spiked
The standard adding recovery rate refers to the ratio of the result obtained by adding quantitative standard substance into the sample matrix without the measured substance and analyzing according to the sample processing steps to the theoretical value.
The Relative Standard Deviation (RSD) is also called standard deviation coefficient, variation coefficient, etc. the precision of the result can be analyzed in the inspection and detection work by dividing the standard deviation by the corresponding average value and multiplying by 100%.
Grinding the reconstituted tobacco into tobacco powder samples, drying to constant weight for later use, weighing 0.2g of the samples by an electronic scale for three times, respectively placing the samples into corresponding reactors, accurately adding 50mL of deionized water, respectively adding 4.12mg, 7.38 mg and 14.26 mg of light calcium carbonate into three groups of reactors, measuring by an atomic absorption spectrophotometer, taking an average value after measuring for 6 times in each group, and substituting into a standard curve to obtain corresponding Ca2+The concentrations, the results of the measurements are shown in Table 2 below.
TABLE 2 recovery of calcium carbonate from reconstituted tobacco with normalized ratio (n =6)
As can be seen from Table 2, the average recovery rate of the three samples is 92.72% -96.61%, which indicates that the reliability of the standard curve in calculating the concentration is high. The relative standard deviation is between 2.83% and 3.52%. The accuracy of the standard curve in calculating the concentration is higher.
Example 2
Ca in blank sample2+Concentration C0The calculation of (2):
s1, grinding the reconstituted tobacco A into tobacco powder, and drying to constant weight for later use;
s2, weighing 0.2g of tobacco powder prepared in the step S1, placing the tobacco powder in a reactor, adding 50mL of deionized water, adding 0.1mg of carbonic anhydrase, sealing the reactor after the reaction is finished, placing the reactor in an ice bath, oscillating for 30min, and then filtering to obtain a blank liquid to be detected;
S3diluting the blank solution to be detected obtained in the step S2 by ice water by 100 times, and measuring Ca by using an atomic absorption spectrophotometer2+Then the absorbance is brought into a standard working curve to obtain Ca in the solution to be detected2+Concentration C of0；
The reconstituted tobacco A in the sample is divided into 6 parts for calcium carbonate content test, and the specific test process is shown in examples 3-8.
Example 3
S1, grinding the reconstituted tobacco A into tobacco powder, and drying to constant weight for later use;
s2, weighing 0.2g of tobacco powder prepared in the step S1, placing the tobacco powder in a reactor, adding 50mL of deionized water, adding 0.1mg of carbonic anhydrase, and introducing CO at the flow rate of 0.5mL/min2Rapidly sealing the container after 20min, placing in ice bath at 0 deg.C, oscillating, and filtering to obtain solution to be detected;
s3, diluting the solution to be detected obtained in the step S2 by 100 times with ice water, and measuring Ca by using an atomic absorption spectrophotometer2+Then the absorbance is brought into a standard working curve to obtain Ca in the solution to be detected2+Concentration C of1；
S4, calculating the content of calcium carbonate in the tobacco leaves:
example 4
S1, grinding the reconstituted tobacco A into tobacco powder, and drying to constant weight for later use;
s2, weighing 0.24g of tobacco powder prepared in the step S1, placing the tobacco powder into a reactor, adding 60mL of deionized water, then adding 0.1mg of carbonic anhydrase, and then introducing CO at the flow rate of 0.8mL/min2Rapidly sealing the container after 30min, placing in ice bath at 0 deg.C, oscillating, and filtering to obtain solution to be detected;
s3, diluting the solution to be detected obtained in the step S2 by 100 times with ice water, and measuring Ca by using an atomic absorption spectrophotometer2+Then the absorbance is brought into a standard working curve to obtain Ca in the solution to be detected2+Concentration C of2；
S4, calculating the content of calcium carbonate in the tobacco leaves:
example 5
S1, grinding the reconstituted tobacco A into tobacco powder, and drying to constant weight for later use;
s2, weighing 0.28g of tobacco powder prepared in the step S1, placing the tobacco powder into a reactor, adding 70mL of deionized water, adding 0.1mg of carbonic anhydrase, and introducing CO at the flow rate of 1.2mL/min2Quickly closing the container after 40min, placing in an ice bath at 0 ℃ for oscillation, and then filtering to obtain a solution to be detected;
s3, diluting the solution to be detected obtained in the step S2 by 100 times with ice water, and measuring Ca by using an atomic absorption spectrophotometer2+Then the absorbance is brought into a standard working curve to obtain Ca in the solution to be detected2+Concentration C of3；
S4, calculating the content of calcium carbonate in the tobacco leaves:
example 6
S1, grinding the reconstituted tobacco A into tobacco powder, and drying to constant weight for later use;
s2, weighing 0.32g of tobacco powder prepared in the step S1, putting the tobacco powder into a reactor, adding 80mL of deionized water, adding 0.1mg of carbonic anhydrase, and introducing CO at the flow rate of 1.5mL/min2Rapidly sealing the container after 50min, placing in ice bath at 0 deg.C, oscillating, and filtering to obtain solution to be detected;
s3, diluting the solution to be detected obtained in the step S2 by 100 times with ice water, and measuring Ca by using an atomic absorption spectrophotometer2+Then the absorbance is brought into a standard working curve to obtain Ca in the solution to be detected2+Concentration C of4；
S4, calculating the content of calcium carbonate in the tobacco leaves:
example 7
S1, grinding the reconstituted tobacco A into tobacco powder, and drying to constant weight for later use;
s2, weighing 0.36g of tobacco powder prepared in the step S1, placing the tobacco powder into a reactor, adding 90mL of deionized water, adding 0.1mg of carbonic anhydrase, and introducing CO at the flow rate of 1.8mL/min2Rapidly closing the container after 60min, placing in an ice bath at 4 ℃ for oscillation, and then filtering to obtain a solution to be detected;
s3, diluting the solution to be detected obtained in the step S2 by 100 times with ice water, and measuring Ca by using an atomic absorption spectrophotometer2+Then the absorbance is brought into a standard working curve to obtain Ca in the solution to be detected2+Concentration C of5；
S4, calculating the content of calcium carbonate in the tobacco leaves:
example 8
S1, grinding the reconstituted tobacco A into tobacco powder, and drying to constant weight for later use;
s2, weighing 0.4g of tobacco powder prepared in the step S1, placing the tobacco powder into a reactor, adding 100mL of deionized water, then adding 0.1mg of carbonic anhydrase, and then introducing CO at the flow rate of 2.0mL/min2Rapidly closing the container after 60min, placing in an ice bath at 4 ℃ for oscillation, and then filtering to obtain a solution to be detected;
s3, diluting the solution to be detected obtained in the step S2 by 100 times with ice water, and measuring Ca by using an atomic absorption spectrophotometer2+Then the absorbance is brought into a standard working curve to obtain Ca in the solution to be detected2+Concentration C of6；
S4, calculating the content of calcium carbonate in the tobacco leaves:
the results of testing the calcium carbonate content of examples 3-8 are shown in table 3 below.
TABLE 3-determination of calcium carbonate in reconstituted tobacco of examples 3-8
As can be seen from Table 3, the errors in the calcium carbonate contents determined in examples 3 to 8 are small, indicating the reliability and accuracy of the method.
The above-described aspects may be implemented individually or in various combinations, and such variations are within the scope of the present invention.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the specific embodiments of the invention be limited to these descriptions. For those skilled in the art to which the invention pertains, numerous simple deductions or substitutions may be made without departing from the spirit of the invention, which shall be deemed to belong to the scope of the invention.
Claims (6)
1. A method for determining calcium carbonate in reconstituted tobacco by applying enzymatic reaction is characterized by comprising the following steps:
s1, grinding the reconstituted tobacco into tobacco powder, and drying to constant weight for later use;
s2, weighing the tobacco powder prepared in the step S1, putting the tobacco powder into a reactor, adding deionized water, adding carbonic anhydrase, and introducing CO2Carrying out reaction, sealing the reactor after the reaction is finished, placing the reactor in an ice bath for oscillation, and then filtering to obtain a solution to be detected;
s3, diluting the solution to be detected obtained in the step S2 with ice water, and measuring Ca by using an atomic absorption spectrophotometer2+Then the absorbance is brought into a standard working curve to obtain Ca in the solution to be detected2+The concentration of (3) C;
the manufacturing method of the standard working curve comprises the following steps: preparing Ca with different concentrations2+Standard solution, measuring Ca with different concentrations by atomic absorption spectrophotometer2+Then Ca2+The concentration is an abscissa, and a standard working curve is drawn by taking the corresponding absorbance as an ordinate;
s4, Ca in blank sample2+Concentration C0The calculating method of (2): weighing the tobacco powder prepared in the step S1, placing the tobacco powder into a reactor, adding deionized water, adding carbonic anhydrase for reaction, sealing the reactor after the reaction is finished, placing the reactor into an ice bath for oscillation, then filtering to obtain a blank liquid to be detected, diluting the blank liquid to be detected with ice water, and measuring Ca by using an atomic absorption spectrophotometer2+Then bringing the absorbance into a standard working curve to obtain Ca in the blank solution to be tested2+Concentration C of0；
S5, calculating the content of calcium carbonate in the reconstituted tobacco according to the following formula:
in the above formula:
x represents the content of calcium carbonate in the reconstituted tobacco, and the unit is microgram per gram (mu g/g);
c-concentration of target in micrograms per milliliter (. mu.g/mL) from the standard working curve;
C0-target concentration in micrograms per milliliter (μ g/mL) in a blank sample from a standard working curve;
v-volume of extract in milliliters (mL);
f is the dilution factor;
m-sample mass in grams (g);
100——CaCO3the formula (2);
40——Ca2+the formula (2).
2. The method for determining calcium carbonate in reconstituted tobacco by using enzymatic reaction as claimed in claim 1, wherein the mass ratio of tobacco powder, deionized water and carbonic anhydrase in steps S2 and S4 is (2000-4000): (500000-1000000): 1.
3. The method for determining calcium carbonate in reconstituted tobacco by using enzymatic reaction according to claim 1, wherein CO is introduced in step S22The time is 20-60 min, and CO is introduced2The flow rate of (A) is 0.5 to 2.0 mL/min.
4. The method for determining calcium carbonate in reconstituted tobacco by using enzymatic reaction according to claim 1, wherein the ice bath temperature in step S2 and step S4 is 0-4 ℃.
5. The method for determining calcium carbonate in reconstituted tobacco by using enzymatic reaction according to claim 1, wherein the shaking time in step S2 and step S4 is 20-60 min.
6. The method for determining calcium carbonate in reconstituted tobacco by using enzymatic reaction according to claim 1, wherein the solution to be determined in step S3 and the blank solution to be determined in step S4 are both diluted by 90-110 times with ice water.
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| CN103675143B (en) * | 2013-12-17 | 2015-08-12 | 红塔烟草(集团)有限责任公司 | The assay method of calcium carbonate content in a kind of reconstituted tobacco |
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