CN108896698B

CN108896698B - Ion chromatography method for simultaneously determining sorbate acid radicals, phosphate radicals and citrate radicals in reconstituted tobacco and application

Info

Publication number: CN108896698B
Application number: CN201810577768.3A
Authority: CN
Inventors: 王予; 赖燕华; 汪军霞; 潘晓薇; 高仁吉
Original assignee: China Tobacco Guangdong Industrial Co Ltd
Current assignee: China Tobacco Guangdong Industrial Co Ltd
Priority date: 2018-06-05
Filing date: 2018-06-05
Publication date: 2020-12-04
Anticipated expiration: 2038-06-05
Also published as: CN108896698A

Abstract

The invention provides an ion chromatography method for simultaneously measuring sorbate acid radicals, phosphate radicals and citrate radicals in reconstituted tobacco. The invention establishes that the column temperature is 23 ℃, 4mmol L^‑1The method for measuring the sorbate acid radicals, the phosphate radicals and the citrate radicals in the reconstituted tobacco by taking the KOH solution AS the initial concentration of the leacheate and an AS11-HC column AS a separation column. The method solves the interference of nitrate radicals to the sorbic acid radicals, ensures that the sorbic acid in the sample is more accurate and reliable in quantitative determination, the relative standard deviation of the tested content of each component to be tested is 0.18-0.74 percent, the standard addition recovery rate is 97.1-102.7 percent, and the method is accurate, simple and reliable. The ion chromatography method is accurate, simple and reliable, and is suitable for simultaneous determination of the sorbate acid radicals, the phosphate radicals and the citrate radicals in the reconstituted tobacco sample.

Description

Ion chromatography method for simultaneously determining sorbate acid radicals, phosphate radicals and citrate radicals in reconstituted tobacco and application

Technical Field

The invention relates to the technical field of detection of reconstituted tobacco, and particularly relates to an ion chromatography method for simultaneously determining sorbate acid radicals, phosphate radicals and citrate radicals in reconstituted tobacco and application of the ion chromatography method.

Background

The paper-making reconstituted tobacco is widely applied to the production process of cigarettes because the paper-making reconstituted tobacco has the effects of balancing the tobacco structure, stabilizing the cigarette quality, effectively reducing the release amount of tar and partial harmful components and the like. Because the reconstituted tobacco leaves use waste tobacco stems, tobacco leaf fragments, tobacco powder and the like as raw materials, the raw materials have certain differences with the tobacco leaves, such as differences in physical properties, sensory quality, tar and harm reduction, functionality and the like, and the quality of the reconstituted tobacco leaves needs to be improved by improvement or covering, one or more additives are added into the reconstituted tobacco leaves to improve one or more properties of the reconstituted tobacco leaves, for example, sorbate is used as a mildew inhibitor; the phosphate is used as an ash content regulator, so that the ash content is firm and not loose; citrate is currently the most commonly used combustion improver. However, the added amount and the residual amount of the additive affect the quality stability and safety of the reconstituted tobacco, so strict control is required.

The ion chromatography has the advantages of good selectivity, high sensitivity, simple pretreatment and the like, and is widely applied to the measurement of anions and cations and organic acids of tobacco and products thereof. However, no report is found at present for simultaneously measuring sorbate, phosphate and citrate in reconstituted tobacco, the reconstituted tobacco has complex components and large ion interference, and the simple pretreatment method cannot remove interfering ions. The following problems exist in the prior determination of the sorbic acid content in reconstituted tobacco: compared with phosphate radical and citrate radical, the sensitivity of the sorbate radical on the separation column is relatively low, the response of the nitrate radical adjacent to the sorbate radical (necessarily existing in the reconstituted tobacco sample) is very high, the interference of the nitrate radical is large, the measured result is inaccurate, and the repeatability is poor.

Disclosure of Invention

The invention aims to establish an ion chromatography method capable of simultaneously measuring sorbate acid radicals, phosphate acid radicals and citrate acid radicals in reconstituted tobacco aiming at the defects of the existing sorbic acid, phosphoric acid and citric acid detection technology in reconstituted tobacco, and provides a technical basis for controlling the addition of chemical substances in the processing process of the reconstituted tobacco.

It is another object of the invention to provide the use of said ion chromatography method.

In order to solve the technical problems, the invention adopts the technical scheme that:

providing an ion chromatography method for simultaneously measuring pyretic acid radicals, phosphate radicals and citrate radicals in reconstituted tobacco, performing oscillation extraction on a reconstituted tobacco sample by using ultrapure water as an extractant, filtering the obtained extract by using a filter membrane to obtain filtrate, and performing ion chromatography analysis;

the chromatographic conditions are as follows: ion exchange column: AS11-HC, 4X 250 mm; column temperature: 23 ℃; sample introduction amount: 50 mu L of the solution; suppressor current: 112 mA; the detector is self-circulation inhibition conductance detection; the leacheate is KOH solution; flow rate: 1.5mL min^-1；

The gradient elution procedure is shown in table 1:

TABLE 1

Preferably, the ion chromatography is preceded by a purification treatment step of the filtrate by using an RP column. Because the components of the reconstituted tobacco are complex, the extract liquid must be purified to remove the components polluting the chromatographic column, and can be subjected to ion chromatography for analysis.

Furthermore, the invention optimizes the packing and filtrate of the RP column, can effectively filter impurity components and has higher pretreatment recovery rate for sorbate, phosphate and citrate.

Preferably, the RP column is an RP column taking polystyrene/divinylbenzene high polymer as a filler.

Preferably, the filtrate further comprises adjusting the pH of the filtrate to 11 prior to performing the purification treatment step.

Preferably, the step of pH adjustment is carried out using a concentration of 250mmol L^-1Is adjusted with NaOH solution.

Preferably, the RP column is activated with 5mL of methanol and 10mL of ultrapure water, respectively, at a flow rate of 1mL/min prior to use.

Preferably, the flow rate of the filtrate in the purification treatment is 0.5 mL/min.

Preferably, the time of the shaking extraction is 30 min.

The three target substances of sorbate, phosphate and citrate can be dissolved in water, and for measuring water-soluble ions, the commonly used extracting agents are pure water, acid solution and alkali solution. As the separation column used in the method is an anion column, a large amount of acid radical ions bring interference, water and NaOH solution are used as extraction liquid, but the extraction efficiency of the phosphate radical is obviously lower under the alkaline condition. Therefore, pure water was chosen as the extractant in this experiment. The invention uses pure water as an extracting agent, and the extraction efficiency of the three target components is the highest when the three target components are oscillated for 20 min.

After careful research and conclusion, the AS11-HC column is preferably used AS the analysis column of the method, and the three target components, namely, the sorbate, the phosphate and the citrate, have relatively good separation effect on the column. However, compared with phosphate radical and citrate radical, the sensitivity of the sorbate radical on the AS11-HC separation column is relatively low, the response of the adjacent nitrate radical is very high, in order to reduce the interference of the nitrate radical on the separation column, the invention further researches and compares the effects of leacheate with different concentrations and column temperature on the separation condition of the sorbate radical and the nitrate radical, the signal-to-noise ratio and the peak shape symmetry of the sorbate radical, and finally determines that the adopted column temperature is 23 ℃, 4mmol L^-1The KOH solution of (1) is the initial concentration of the leacheate, and the chromatographic condition can ensure that the sorbate radical is separated from the nitrate radical to reach a base line with better peak shape and sensitivity.

The ion chromatography method can be well applied to determination of sorbic acid, phosphoric acid and/or citric acid components in reconstituted tobacco. And the ion chromatography method can also be applied to the determination of sorbic acid, phosphoric acid and/or citric acid components in the additives in the reconstituted tobacco. By utilizing the ion chromatography method, the Relative Standard Deviation (RSD) of the tested content of each component to be tested is 0.18-0.74%, the standard addition recovery rate is 97.1-102.7%, and the ion chromatography method is accurate, simple and reliable.

Compared with the prior art, the invention has the beneficial effects that:

the invention fills the blank of the prior art, provides an ion chromatography method for simultaneously measuring the sorbate acid radicals, the phosphate radicals and the citrate radicals in the reconstituted tobacco, and establishes 4mmol L of ion chromatography method with the column temperature of 23 DEG C^-1The method for measuring the sorbate acid radicals, the phosphate radicals and the citrate radicals in the reconstituted tobacco by taking the KOH solution AS the initial concentration of the leacheate and an AS11-HC column AS a separation column. The method solves the problem of interference of nitrate radicals on the sorbate radicals, and ensures that the sorbic acid in the sample is more accurate and reliable in quantification. When the ion chromatography method is applied to determination of sorbic acid, phosphoric acid and citric acid in reconstituted tobacco, the Relative Standard Deviation (RSD) of the content of each component to be determined is 0.18-0.74%, and the standard addition recovery rate is 97.1-102.7%, so that the method is accurate, simple and reliable.

Drawings

FIG. 1 is a chromatogram of an actual sample (wherein 1-sorbate, 2-is nitrate, 3-phosphate, 4-citrate).

FIG. 2 is a chromatogram of a mixed standard solution (1-sorbate, 2-phosphate, 3-citrate).

Detailed Description

The invention is further illustrated by the following specific examples. The following examples are for illustrative purposes only and are not to be construed as limiting the invention. Unless otherwise specified, the reagents used in the following examples are those conventionally commercially available or commercially available, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art.

Materials and methods

1) Device

ICS5000 ion chromatograph (dean, usa); automatic solid phase extractors (Gilson corporation, usa); Milli-Q ultra pure water instruments (Millipore, USA).

2) Material

The invention takes 26 randomly selected reconstituted tobacco test products and finished products as an example for explanation; potassium sorbate (99.5%, sigma, usa); phosphate radical standard solution (1000. mu.g mL)^-1National center for standards materials); citric acid (99%, sigma, usa); ultrapure water (resistivity is more than or equal to 18.2M omega cm); sodium hydroxide solution (50%, Fluka, usa); filters (0.45 μm, Hippo); RP columns (1CC, dean usa); analytical column (AS11-HC 4mm, Daian USA); automatic leacheate (potassium hydroxide, dean, usa).

Example 1 Effect test of extractant

The three target components of the sorbate, the phosphate and the citrate which are measured by the invention can be dissolved in water, and for measuring water-soluble ions, common extracting agents are pure water, acid solution and alkali solution. Since the separation column used in the method was an anion column, and a large amount of acid ions caused interference, it was attempted to use water and 4mmol L^-1NaOH solution as an extraction liquid. The results (see table 2) show that: of phosphates under alkaline conditionsThe extraction efficiency is obviously low. Therefore, pure water is selected as the extracting agent in the experiment.

TABLE 2 comparison of extraction efficiencies of different extractants

The content of the target component was determined for the same weight of sample at different extraction times using pure water as the extractant (see table 3). The test shows that: pure water is used as an extracting agent, and when the three target components are oscillated for 20min, the extraction efficiency of the three target components is the highest, so that water is used as the extracting agent, and the extraction time is 20 min. Meanwhile, the residue after 20min of extraction is subjected to secondary extraction, and the target component cannot be detected, which indicates that the primary extraction is complete.

TABLE 3 concentration of extracted target component at different extraction times

Example 2 research experiment of sample solution purification treatment

Because the components of the reconstituted tobacco are complex, the sample extract liquid can enter the ion chromatography for analysis only by purifying and removing the components which pollute the chromatographic column, and therefore, the experiment investigates the purifying effects of the RP column and the C18 solid-phase extraction column. Because the component to be detected contains an organic group and an ionizable group, ionization is inhibited under neutral and acidic conditions, and a certain retention is realized on the solid phase column, so that the recovery rate after purification is low. The sample extract was applied to 250mmol L^-1The NaOH solution is adjusted to be alkaline (the use amount of the high-concentration alkaline solution can be reduced to achieve the purpose of neglecting volume change), the ionization of acid radical groups can be promoted, the interaction between the acid radical groups and the pretreatment column is weakened, and therefore good recovery rate is obtained. Discomfort due to C18 columnIn the experiment, an RP column which takes polystyrene/divinylbenzene high polymer as a filler is adopted to purify the sample liquid when the pH value of the sample liquid is too high or too low. The results in table 4 show: the pH value has little influence on the recovery rate of phosphate radical and citrate radical, and has great influence on the recovery rate of sorbate radical. By adjusting the pH value of the extracting solution, the experiment confirms that the pretreatment method of adjusting the pH value of the sample extracting solution to 11 and then passing through an RP column is adopted.

TABLE 4 purification recovery at different pH values

Example 3 determination of chromatographic conditions

The experiment examines the peak appearance conditions of the sorbate, the phosphate and the citrate on three ion chromatographic columns of AS20, AS18 and AS11-HC respectively, and through comparison, the separation degree, the peak shape symmetry, the peak width and the sensitivity of the phosphate and the citrate on the three chromatographic columns are better, while the difference of the sorbate is larger, and the result shows that the peak appearance condition of the sorbate on the AS11-HC column is optimal, so the AS11-HC chromatographic column is selected in the experiment.

Compared with phosphate and citrate, the sensitivity of the sorbate on the AS11-HC separation column is relatively low, the response of the adjacent nitrate is high, and in order to reduce the interference of the nitrate on the sorbate, the effects of leacheate with different concentrations and the column temperature on the separation of two acid ions, the signal-to-noise ratio of the sorbate and the symmetry of the peak shape are compared. Finally, the column temperature is determined to be 23 ℃, 4mmol L^-1The chromatographic condition enables the sorbate to achieve baseline separation from the nitrate with better peak shape and sensitivity (see figure 1, in figure 1, peak 1 is sorbate, peak 2 is nitrate, peak 3 is phosphate, and peak 4 is citrate).

In addition, the present test also examined the influence of seven common anions and organic acid groups such as formate, acetate, propionate, lactate, malate, succinate, malonate, oxalate and the like in tobacco on the target component. The anions were found to peak before 16min and within 22-35 min, with good separation from the components to be tested (see FIG. 1).

Since phosphate and citrate are strongly retained ions, gradient elution is adopted in the experiment, and after weakly retained sorbate is eluted, the concentration of the eluent is increased to enable the strongly retained component to be eluted as soon as possible, so that the elution time is shortened (see fig. 2, in fig. 2, peak 1 is sorbate, peak 2 is phosphate, and peak 3 is citrate).

Example 4 determination of working curves and detection limits

Respectively using pure water and 4mmol L^-1The standard solution is prepared from the NaOH solution, and the responses of three acid radical ions are not greatly different under two matrixes (see table 5). To simplify the operation, the standard solution was prepared using pure water. Preparing mixed standard stock solutions of sorbate, phosphate and citrate with the concentrations of 100 mu g mL respectively^-1、1000μg mL^-1And 1000. mu.g mL^-1. Transferring 0.05 mL, 0.25 mL, 0.5mL, 0.75 mL and 1.0mL of the mixed stock solutions into 25mL volumetric flasks, diluting to constant volume with ultrapure water, preparing a series of standard working solutions (see Table 6), performing IC analysis, and determining the corresponding concentrations (X, μ g mL) according to the peak area (Y) of the target^-1) Linear regression was performed. And taking the standard solution with the lowest concentration in the series of standard solutions, continuously injecting samples for 10 times, calculating the Standard Deviation (SD) of data, and taking 3 times of SD as the detection limit of the method. The regression equation, correlation coefficient and detection limit are shown in table 7.

TABLE 5 comparison of the areas of the peaks of the three acid ions under different substrates

TABLE 6 concentration of the mixed standard working solution (. mu.g mL)^-1)

TABLE 7 regression equation, correlation coefficient and detection limits

Example 5 recovery and precision experiments

Three reconstituted tobacco samples are taken and measured according to the following method.

Pretreatment of samples

Samples were prepared and water was measured according to GB/T19616 and YC/T31-1996 by accurately weighing 0.15g of sample (to 0.0001g accuracy) in a 150mL ground flask, adding 50.0mL of ultrapure water, stoppering, placing on a shaker and shaking for 20 min. Filtering the extract with a membrane, and adding 250mmol L^-1The pH of the NaOH solution is adjusted to 11. 5mL of pH 11 sample solution was taken for 0.5mL min^-1The flow rate of (2) was passed through an activated (respectively activated with 5mL of methanol and 10mL of ultrapure water) RP column, the first 3mL of solution was discarded, and the following 2mL of solution was collected for ion chromatography.

Chromatographic conditions

Ion exchange column: AS11-HC (4X 250 mm); column temperature: 23 ℃; leacheate: a KOH solution; flow rate: 1.5mL min^-1(ii) a Gradient elution procedure: 4mmol L^-1(0～23min)，4～30mmol L^-1(24～35min)，30mmol L^-1(36～39min)，4mmol L^-1(40-44 min); sample introduction amount: 50 mu L of the solution; suppressor current: 112 mA. A detector: self-cycling inhibition conductance measurements.

Each sample is repeated for 5 times, and the result shows (see table 8) that the Relative Standard Deviation (RSD) of the content of the target component in the sample is 0.18-0.57%, which indicates that the repeatability of the method is better. Taking a sample solution with known content, respectively adding three standard solutions with concentration levels for IC analysis, and calculating the recovery rate, wherein the result shows (see table 9) that the recovery rate is between 97.1% and 102.7%, which indicates that the method has high accuracy and can meet the requirement of quantitative analysis.

Table 8 method repeatability (n ═ 5)

Table 9 recovery of three acid ions in the sample

EXAMPLE 6 actual measurement of sample

Pretreatment of samples

Chromatographic conditions

The results of the determination of 26 reconstituted tobacco samples by adopting the method are shown in Table 10, wherein the mass fraction of the sorbate in the reconstituted tobacco is 5.5-741.2 mu g g^-1The mass fraction of the phosphate radical is 59.3-6977.9 mu g g^-1The mass fraction of the citrate is 259.2-9642.7 mu g g^-1。

TABLE 10 content of three acid ions in the actual samples

The invention establishes a method for measuring the contents of sorbate acid radicals, phosphate radicals and citrate radicals in reconstituted tobacco by using an ion chromatography method with an RP column AS a purification column and AS11-HC AS a separation column. The method is simple, accurate and reliable, has good repeatability, and is suitable for evaluating the residual quantity of the three substances in the process of manufacturing the reconstituted tobacco. In addition, the method is also suitable for the determination of tobacco.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An ion chromatography method for simultaneously determining sorbate acid radicals, phosphate acid radicals and citrate acid radicals in reconstituted tobacco is characterized in that a reconstituted tobacco sample is subjected to oscillation extraction by taking ultrapure water as an extractant, obtained extract is filtered by a filter membrane to obtain filtrate, and then ion chromatography analysis is carried out;

The gradient elution procedure was:

before the ion chromatography, purifying the filtrate by using an RP column; the RP column is an RP column taking a polystyrene/divinylbenzene high polymer as a filler; the filtrate further comprises adjusting the pH of the filtrate to 11 prior to performing the purification treatment step.

2. The ion chromatography method of claim 1, wherein the step of adjusting the pH is performed with a concentration of 250mmol L^-1Is adjusted with NaOH solution.

3. Use of the ion chromatography method according to claim 1 or 2, characterized in that it is used for the determination of the sorbic acid, phosphoric acid and/or citric acid component in reconstituted tobacco.

4. Use of the ion chromatography method according to claim 1 or 2, characterized in that it is used for the determination of sorbic acid, phosphoric acid and/or citric acid components in additives in reconstituted tobacco.