CN115993408A - Method for measuring four organic acids in tobacco flavor and fragrance - Google Patents
Method for measuring four organic acids in tobacco flavor and fragrance Download PDFInfo
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- CN115993408A CN115993408A CN202211363233.9A CN202211363233A CN115993408A CN 115993408 A CN115993408 A CN 115993408A CN 202211363233 A CN202211363233 A CN 202211363233A CN 115993408 A CN115993408 A CN 115993408A
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Abstract
The invention belongs to the technical field of tobacco chemical analysis, and particularly discloses a method for measuring four organic acids of citric acid, malic acid, lactic acid and acetic acid in tobacco essence and spice. The method adopts ultrapure water as an extraction solvent, utilizes vortex oscillation-RP small columns to finish sample pretreatment, and detects organic acid components in the sample by ion exclusion chromatography qualitative analysis, and has the advantages of high precision, wide applicability and capability of detecting more components of a complex system sample.
Description
Technical Field
The invention belongs to the technical field of tobacco chemical analysis, and particularly relates to a method for measuring contents of citric acid, malic acid, lactic acid and acetic acid in tobacco essence and spice.
Background
The essence perfume is a general name of aroma substances, and is used as one of cigarette additives, and mainly has the effects of enhancing the aroma of tobacco products, improving the taste and shaping unique product styles. Its quality stability is directly related to the quality stability of the cigarette product. In order to achieve the aim of quality control, the tobacco flavor and fragrance is usually measured by adopting a method with larger physical and chemical indexes of sensory evaluation and acidity and subjective factors, and a certain subjective error exists. Therefore, it is necessary to perform analysis objectively by using a chemical analysis method.
At present, a gas chromatography mass spectrometry method is mostly adopted for testing organic acid ions in the essence and the spice for the cigarettes. However, the gas quality method is relatively complicated, long-time derivatization (for example, 24 h) extraction quantitative detection is required according to literature reports, and the efficiency is poor when the gas quality method is practically used for sample screening.
Aiming at the analysis problem of the organic acid in the existing tobacco flavor and fragrance, the invention aims to realize the purpose of accurately measuring the organic acid in the tobacco flavor and fragrance by optimizing and adjusting instrument parameters and conditions.
Disclosure of Invention
The invention aims to establish a method for measuring citric acid, malic acid, lactic acid and acetic acid in essence and spice with high sensitivity, accuracy, rapidness, stability and anti-interference capability, and the method can realize perfect separation of organic acid with polarity close to that in the essence and spice and has the advantages of simple pretreatment, stable peak time and accuracy and rapidness in measurement.
The invention aims at realizing the following technical scheme:
the method for measuring four organic acids in the essence and the spice specifically comprises the following steps:
first, standard solution preparation: respectively weighing citric acid, malic acid, lactic acid and acetic acid standard substances to prepare mixed standard mother liquor, and carrying out gradient dilution when carrying out linear measurement on organic acid;
secondly, sample pretreatment: taking essence and spice for cigarettes, taking a sample, placing the sample into a centrifuge tube, accurately adding quantitative ultrapure water, and carrying out vortex oscillation;
third, onGuard RP column activation: passing through methanol and ultrapure water respectively;
fourth, preparing a sample to-be-detected liquid: filtering the vortex-oscillated test solution into a chromatographic bottle by using an activated OnGuard RP small column and a 0.45 aqueous phase filter membrane to obtain a solution to be tested;
fifth step, organic acid characterization: and (3) respectively detecting the standard solution and the liquid to be detected by adopting an ion exclusion chromatography on-machine, drawing four organic acid standard curves according to the peak area obtained by integrating the standard solution, and calculating to obtain the contents of the four organic acids in the liquid to be detected.
Preferably, the standard mother liquor and the ultrapure water are mixed in the first step, the mixed standard mother liquor is subjected to gradient dilution, and the concentration range of each organic acid is 0.5-20mg/L.
Preferably, in the second step, the dosage of the essence and spice sample for the cigarettes is 0.5g, the dosage of the ultrapure water is 50mL, the vortex oscillation time is 10min, and the specification of the centrifuge tube is 50mL.
Preferably, in the third step, the volumes of methanol and ultrapure water used are 10mL.
Preferably, in the fourth step, the first 3mL of the reagent that was retained during RP column activation is discarded when the OnGuard RP column was used for filtration.
Preferably, in the fifth step, the ion exclusion chromatography is performed under the following conditions: the chromatographic column is an anion exclusion chromatographic column IonPac ICE AS6; the suppressor is AMMS ICE300; the detector is a conductivity detector; the eluent is 0.4mM of heptafluorobutyric acid solution; the regeneration solution is 5mM tetrabutylammonium hydroxide solution; column temperature 19 ℃; the flow rate is 1.0mL/min; the sample volume was 50uL.
The invention examines and optimizes instrument parameters and conditions affecting detection results, and establishes a method for measuring citric acid, malic acid, lactic acid and acetic acid in essence and spice with high sensitivity, accuracy, rapidness, stability and anti-interference capability.
Drawings
FIG. 1 is a graph of a standard solution for testing an AS11-HC column in example 1 of the present invention.
FIG. 2 is a graph of a sample solution to be tested using an AS11-HC column in example 1 of the present invention.
FIG. 3 is a graph of a standard solution for testing an AS19 column according to example 2 of the present invention.
FIG. 4 is a graph of a sample fluid to be tested using an AS19 column according to example 2 of the present invention.
FIG. 5 is a graph of a standard solution for the ICE AS6 exclusion column test of example 3 of the present invention.
FIG. 6 is a graph of the test sample fluid under test using ICE AS6 exclusion column according to example 3 of the present invention.
Fig. 7 is an ICE AS6 exclusion column test chart of the test solution of the tobacco flavor sample 1 according to example 4 of the present invention.
Fig. 8 is an ICE AS6 exclusion column test chart of the test solution of the tobacco flavor sample 2 according to example 4 of the present invention.
Fig. 9 is an ICE AS6 exclusion column test chart of the sample liquid 3 of the essence for cigarettes of example 4 of the present invention.
Detailed Description
The invention is further described in connection with the following detailed description.
The invention examines and optimizes the instrument parameters and conditions affecting the detection result. In order to obtain a better separation spectrogram effect, the invention examines the influence of the type of chromatographic column and the parameter conditions of the instrument on the content of four organic acids in the essence and spice sample.
Example 1: AS11-HC chromatographic column for measuring essence and spice sample 1
(1) Standard solution preparation
Respectively weighing citric acid, malic acid, lactic acid and acetic acid standard substances to prepare mixed standard mother liquor, carrying out gradient dilution when carrying out linear measurement on organic acid, wherein ultrapure water is used for the mixed standard mother liquor and the gradient dilution, and the concentration range of each organic acid is 0.5-20mg/L;
(2) Preparation of essence and spice sample 1 to-be-detected liquid
Accurately measuring 0.5g of tobacco essence and spice sample 1, adding 50mL of ultrapure water, vortexing for 10min, filtering by using an activated (activated is respectively carried out by using 10mL of methanol and 10mL of ultrapure water to pass through a column) OnGuard RP small column, and discarding the previous 3mL of solution to prepare the essence and spice sample 1 to-be-detected liquid.
(3) Chromatographic conditions
Chromatographic column: dionex IonPac AS11-HC (250 mm. Times.4 mm);
protective column: dionex IonPac AG11-HC (50 mm. Times.4 mm);
mobile phase: potassium hydroxide solution;
leaching gradient: the concentration of the potassium hydroxide solution is 0-9min,1mmol/L;9-17min,1-13mmol/L;17-27min,13mmol/L;27-29min,13-45mmol/L;29-32min,45mmol/L;32.1min,1mmol/L;32.1-35min,1mmol/L;
flow rate: 1.2mL/min;
a suppressor: ARDS600 anion suppressor;
a detector: a conductivity detector;
column temperature: 30 ℃;
sample injection volume: 25uL.
(4) Quantitative determination
And (3) respectively taking the prepared standard solution sample and the sample 1 to-be-detected solution, performing chromatographic detection by adopting the chromatographic conditions, and recording chromatograms, such as the figures 1 and 2.
As shown in figures 1 and 2, the AS11-HC column detection standard solution and the essence and perfume sample 1 have better peak type, but the separation degree of lactic acid and acetic acid is less than 1.5, and the determination is difficult.
Example 2: AS19 chromatographic column for measuring essence and spice sample 1
(1) Standard solution preparation
Respectively weighing citric acid, malic acid, lactic acid and acetic acid standard substances to prepare mixed standard mother liquor, carrying out gradient dilution when carrying out linear measurement on organic acid, wherein ultrapure water is used for the mixed standard mother liquor and the gradient dilution, and the concentration range of each organic acid is 0.5-20mg/L;
(2) Preparation of essence and spice sample 1 to-be-detected liquid
Accurately weighing 0.5g of essence and spice sample 1 for cigarettes, adding 50mL of ultrapure water, vortexing for 10min, filtering by using an activated OnGuard RP small column (the activation is carried out by using 10mL of methanol and 10mL of ultrapure water respectively), and discarding the previous 3mL of solution to prepare the essence and spice sample 1 to-be-detected liquid.
(3) Chromatographic conditions
Chromatographic column: dionex IonPac AS19 (250 mm. Times.4 mm);
protective column: dionex IonPac AG19 (50 mm. Times.4 mm);
mobile phase: potassium hydroxide solution;
leaching gradient: the concentration of the potassium hydroxide solution is 0-15min,2mmol/L;15-30min,2-40mmol/L;30-40min,40mmol/L;40-43min,2mmol/L;
flow rate: 1.0mL/min;
a suppressor: ARDS600 anion suppressor;
a detector: a conductivity detector;
column temperature: 30 ℃;
sample injection volume: 25uL.
(4) Quantitative determination
And (3) respectively taking the prepared standard solution sample and the sample 1 to-be-detected solution, performing chromatographic detection by adopting the chromatographic conditions, and recording chromatograms, such as fig. 3 and 4.
As shown in figures 3 and 4, the AS19 column detection standard solution and the essence and perfume sample 1 have better peak type, but the separation degree of lactic acid and acetic acid still can not reach 1.5, and the accurate quantification can not be realized.
Example 3: determination of essence and perfume sample 1 by exclusion chromatography column ICE AS6 chromatography column
(1) Standard solution preparation
Respectively weighing citric acid, malic acid, lactic acid and acetic acid standard substances to prepare mixed standard mother liquor, carrying out gradient dilution when carrying out linear measurement on organic acid, wherein ultrapure water is used for the mixed standard mother liquor and the gradient dilution, and the concentration range of each organic acid is 0.5-20mg/L;
(2) Preparation of essence and spice sample to-be-detected liquid
Accurately weighing 0.5g of essence and spice sample 1 for cigarettes, adding 50mL of ultrapure water, vortexing for 10min, filtering by using an activated OnGuard RP small column (the activation is carried out by using 10mL of methanol and 10mL of ultrapure water respectively), and discarding the previous 3mL of solution to prepare the essence and spice sample 1 to-be-detected liquid.
(3) Chromatographic conditions
Chromatographic column: dionex IonPac ICE AS6;
the suppressor is AMMS ICE300;
the detector is a conductivity detector;
the eluent is 0.4mM of heptafluorobutyric acid solution;
the regeneration solution is 5mM tetrabutylammonium hydroxide solution;
column temperature 19 ℃;
the flow rate is 1.0mL/min;
the sample volume was 50uL.
(4) Quantitative determination
And (3) respectively taking the prepared standard solution sample and the sample 1 to-be-detected solution, performing chromatographic detection by adopting the chromatographic conditions, and recording chromatograms, as shown in fig. 5 and 6.
As shown in the results of fig. 5 and 6, the exclusion column has the front sequence of citric acid, malic acid, lactic acid and acetic acid, the front time of the test sample can correspond to that of the standard solution, the peak type is good, and the separation degree can reach 1.5.
Example 4: determination of essence and perfume sample by using exclusion chromatography column ICE AS6 chromatography column
(1) Standard solution preparation
Respectively weighing citric acid, malic acid, lactic acid and acetic acid standard substances to prepare mixed standard mother liquor, carrying out gradient dilution when carrying out linear measurement on organic acid, wherein ultrapure water is used for the mixed standard mother liquor and the gradient dilution, and the concentration range of each organic acid is 0.5-20mg/L;
(2) Preparation of essence and spice sample to-be-detected liquid
0.5g of essence and spice sample 1 (feed liquid), sample 2 (essence) and sample 3 (material block) for cigarettes are respectively and accurately weighed, 50mL of ultra-pure water is respectively added, vortex oscillation time is 10min, an activated (the activation is respectively carried out by 10mL of methanol and 10mL of ultra-pure water, and the column is carried out by column filtration) OnGuard RP small column, and the previous 3mL of solution is discarded, so that the essence and spice sample 1, sample 2 and sample 3 to-be-detected liquid are prepared.
(3) Chromatographic conditions
Chromatographic column: dionex IonPac ICE AS6;
the suppressor is AMMS ICE300;
the detector is a conductivity detector;
the eluent is 0.4mM of heptafluorobutyric acid solution;
the regeneration solution is 5mM tetrabutylammonium hydroxide solution;
column temperature 19 ℃;
the flow rate is 1.0mL/min;
the sample volume was 50uL.
(4) Quantitative determination
The prepared sample 1, sample 2 and sample 3 to-be-detected liquid are respectively taken, chromatographic detection is carried out by adopting the chromatographic conditions, and chromatograms are recorded, as shown in fig. 7-9, the detection of essence and spice samples with different forms by excluding chromatographic columns can be seen, and four organic acids can be well separated and detected.
(5) Linear range and detection limit
And (3) carrying out chromatographic analysis on the mixed standard solution prepared from the four organic acid standards, and carrying out correlation coefficient and linear regression analysis on the gradient values. The results show that the correlation coefficient of four organic acids is 0.9999, which shows that the linear correlation of the peak area and the mass concentration of each organic acid component is good, and the organic acid content can be well measured under the condition. When the signal-to-noise ratio was 3, the detection limit of each of the four organic acids was 0.1mg/L.
(6) Accuracy investigation
The mixed standard solution is continuously sampled for 7 times, and RSD of citric acid, malic acid, lactic acid and acetic acid are respectively 0.2%, 0.3%, 0.2% and 0.4% according to the obtained peak area investigation accuracy, which shows that the method has good accuracy.
(7) Repeatability investigation
7 parts of uniformly mixed essence and spice samples are taken, continuous sample injection is carried out, and RSD of citric acid, malic acid, lactic acid and acetic acid are measured to be 0.30%, 0.35%, 0.27% and 0.32% respectively, which shows that the repeatability of the method can meet the analysis requirement.
Therefore, the method has the advantages of complete separation of organic acid ions, simple pretreatment, stable peak time and accurate and rapid determination.
Claims (6)
1. The method for measuring four organic acids of citric acid, malic acid, lactic acid and acetic acid in the essence and the spice for the cigarettes is characterized by comprising the following steps of:
first, standard solution preparation: respectively weighing citric acid, malic acid, lactic acid and acetic acid standard substances to prepare mixed standard mother liquor, and carrying out gradient dilution when carrying out linear measurement on organic acid;
secondly, sample pretreatment: taking essence and spice for cigarettes, taking a sample, placing the sample into a centrifuge tube, accurately adding quantitative ultrapure water, and carrying out vortex oscillation;
third, onGuard RP column activation: passing through methanol and ultrapure water respectively;
fourth, preparing a sample to-be-detected liquid: filtering the vortex-oscillated test solution into a chromatographic bottle by using an activated OnGuard RP small column and a 0.45 aqueous phase filter membrane to obtain a solution to be tested;
fifth step, organic acid characterization: and (3) respectively detecting the standard solution and the liquid to be detected by adopting an ion exclusion chromatography on-machine, drawing four organic acid standard curves according to the peak area obtained by integrating the standard solution, and calculating to obtain the contents of the four organic acids in the liquid to be detected.
2. The assay of claim 1, wherein: in the first step, the standard mother solution and the ultrapure water are mixed for gradient dilution, and the mixed standard mother solution is subjected to gradient dilution, wherein the concentration range of each organic acid is 0.5-20mg/L.
3. The assay of claim 1, wherein: in the second step, the dosage of the tobacco essence and spice sample is 0.5g, the dosage of ultrapure water is 50mL, the vortex oscillation time is 10min, and the specification of a centrifuge tube is 50mL.
4. The assay of claim 1, wherein: in the third step, the volumes of methanol and ultrapure water used were 10mL.
5. The assay of claim 1, wherein: in the fourth step, onGuard RP is adopted
The first 3mL of the reagent that was retained during RP column activation was discarded during column filtration.
6. The assay of claim 1, wherein: in the fifth step, the conditions of the ion exclusion chromatography are as follows: the chromatographic column is an anion exclusion chromatographic column IonPac ICE AS6; the suppressor is AMMSICE300; the detector is a conductivity detector; the eluent is 0.4mM of heptafluorobutyric acid solution; the regeneration solution is 5mM tetrabutylammonium hydroxide solution; column temperature 19 ℃; the flow rate is 1.0mL/min; the sample volume was 50uL.
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| CN117816388A (en) * | 2024-03-05 | 2024-04-05 | 中储粮成都储藏研究院有限公司 | Grain impurity cleaning method and system |
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