CN101071104B - Method for Determination of Chlorine Content in Tobacco - Google Patents

Abstract

The invention discloses a method for measuring chlorine content in tobacco, which provides suitable conditions for measuring chlorine content in tobacco by continuous flow analysis, thereby obtaining the most suitable method for measuring chlorine content in tobacco, which is not only compatible with Mohr's method, potential The detection results of classic analysis methods such as titration and ion chromatography have a high degree of consistency, and compared with the existing continuous flow analyzer test efficiency, the sample detection cycle is shortened by nearly 40%, which is a simple, fast and easy method. It is a measurement method with accurate operation and detection results and high reliability.

Description

Determination method of chlorine content in tobacco

technical field

The invention relates to an analysis method, in particular to a continuous flow analysis method for chlorine content in tobacco. the

Background technique

Chlorine content is closely related to the quality of tobacco, and it is an important technical index in the comprehensive quality evaluation of tobacco and its products. In recent years, many domestic tobacco companies have used continuous flow analyzers to measure the chlorine content in tobacco. The basic principle of the continuous flow analysis method is to release thiocyanate radicals through the reaction of chlorine and mercury thiocyanate, and then complex with ferric iron to develop color, and then use a photometer for measurement, which is actually a spectrophotometric method. The specific operation method is to extract samples according to the method stipulated in "GB/T5606.1-1996", prepare tobacco powder samples according to "YC/T31-1996", and do moisture content experiment at the same time. Weigh about 0.5g (accurate to 0.1mg) of tobacco powder, place in 100mL of 5% (volume fraction) acetic acid, shake and extract for 30min, then filter with qualitative filter paper, and the filtrate is measured with an automatic analyzer. Commonly used automatic analyzers and detection methods are: German AAII/AAIII, Dutch SKALAR, French ALLIANCE, American API, YC/T162-2002 and GB13580.9-92. The principles of these instruments and methods for detecting chlorine are basically the same. Its specific workflow can refer to Figure 1 - the current method for measuring chlorine content in tobacco by flow analysis method in China Tobacco Industry Standard. the

The entire operation process including extraction and instrumental spectrophotometric analysis is carried out in an ordinary air-conditioned environment with a temperature of 20°C-30°C. the

However, the spectrophotometric analysis of the instrument is affected by many factors. For the detection of chlorine content in tobacco, the detection results of continuous flow analysis method and classical analysis methods such as Mohr method, potentiometric titration method, ion chromatography, etc. are quite different. big difference. the

Through several experiments and summaries, the applicant has concluded that the continuous flow analysis method adopts dialysis treatment, 37°C color development and 450-490nm detection wavelength are the suitable conditions for the continuous flow analysis method to measure the chlorine content of tobacco, and writes related papers, according to Further studies have found that the accuracy and rapidity of the results of continuous flow analysis are still interfered by other related factors. the

Continuous flow analysis technology has the advantages of simplicity, quickness, and easy operation. If a more systematic measurement method can be found and applied to continuous flow analysis instruments, continuous flow analysis technology can have the advantages of accurate detection results and high reliability. It will have a wider application in the tobacco industry, and it is bound to become the method followed by the manufacturers of related instruments. the

Contents of the invention

The object of the present invention is to provide a method for measuring chlorine content in tobacco aiming at the deficiency of continuous flow analysis technology. the

The technical solution of the present invention is to provide a method for measuring the chlorine content in tobacco. The chlorine reacts with mercury thiocyanate to release thiocyanate, and then complexes with ferric iron to develop color, and then measures it by spectrophotometry. Dialysis treatment, color development at 37°C, detection wavelength at 450-490nm, and color reaction time of 660-670 seconds are used for photometric determination, especially through the use of 485nm±4nm detection wavelength, adjustment of dialysis solvent, color development agent and mainstream The flow rate ratio of the solvent, the sample dilution ratio, and the combination of the colorimetric cell with appropriate specifications can obtain more accurate and rapid measurement results. the

The preferred detection wavelength of the present invention is 485nm. the

The colorimetric cell of the present invention adopts the 10mm non-foaming colorimetric cell, cooperates and adopts 1M nitric acid solution as the dialysis solvent, water as the main solvent, and the flow rate ratio of the color developer and the mainstream solvent is 1.20 milliliters/minute: 1.00 milliliters/minute Minutes, the dilution ratio is 0.80 ml/min: 0.32 ml/min using the ratio of sample volume to dialysis solvent flow rate. the

The instrument response value of the 10mm non-foaming cuvette is similar to that of the common 10mm cuvette, but it is much less affected by the diffusion effect of the solute, so its detection time period is also the shortest. The present invention adopts the 10mm non-foaming cuvette. the

But on the other hand, the use of a 10mm colorimetric cell with a small optical path length will cause a decrease in the sensitivity of the instrument and affect the detection of tobacco samples with low chloride ion content. Therefore, 1M nitric acid solution was used as the dialysis solvent and water as the main solvent. And adjust the flow rate ratio of the chromogenic solvent and the mainstream solvent to be 1.20 ml/min: 1.00 ml/min, and the flow ratio of the sample dilution ratio injection volume to the dialysis solvent to be 0.32 ml/min: 0.80 ml/min. the

The beneficial effect of the present invention is to limit the suitable condition of the continuous flow analysis method to measure the chlorine content in the tobacco, thereby obtain the most suitable method of measuring the chlorine content in the tobacco, not only with the classic methods such as Mohr method, potentiometric titration method, ion chromatography The detection results of the analytical method have a high degree of consistency—RSD<3%, t<t _1-a=0.10/2 (n-1), and the sample detection cycle is shortened by nearly 40% compared with before adjustment, which is a A simple, quick, easy-to-operate, accurate test result, high reliability determination method.

Description of drawings

Figure 1 Reference diagram of the flow analysis method for the determination of chlorine content in China's current tobacco industry standard

The chromaticity diagram of the standard solution after Fig. 2 embodiment 1 sample extract of the present invention and color development

Fig. 3 is the accompanying drawing of the absorbance of the different sample extracts of Table 1 in Example 1 of the present invention

The chromaticity diagram of the sample extract of Fig. 4 embodiment of the present invention 1 different treatment modes

The chromaticity diagram of the sample extract after the dialysis treatment of Fig. 5 embodiment of the present invention 2

The sample chromaticity figure of Fig. 6 embodiment of the present invention 3 different color developing reaction times

The absorbance spectrogram when Fig. 7 embodiment 4 of the present invention different optical paths

Detailed ways

Figure 8 is a reference diagram of the workflow of the assay method of the present invention

The present invention will be further described in detail below in conjunction with specific experimental data and examples. the

The selection experiment of embodiment 1 dialysis treatment

1. Instruments, samples and main reagents Skalar SAN ⁺⁺ continuous flow analyzer (Netherlands, SKALAR), Lambda850 UV-visible spectrophotometer (USA, PE), H110 electronic balance (Germany, Sartorius, Sensitivity : 0.1mg); cross-linked aromatic polyamide composite semipermeable membrane (SKALAR-5283), polyethoxylauryl ether (30%, Skalar, Netherlands), mercury thiocyanate, methanol, ferric nitrate and nitric acid, all analyzed Pure reagents. Standard tobacco leaf samples GBW08514 and GBW08515 (Qingzhou Tobacco Research Institute).

2. Sample processing and analysis

Take samples according to the method stipulated in "GB/T5606.1-1996", prepare tobacco powder samples according to "YC/T31-1996", and do moisture content experiment at the same time. Weigh about 0.5g (accurate to 0.1mg) of tobacco powder, place in 100mL of 5% (volume fraction) acetic acid, shake and extract for 30min, then filter with qualitative filter paper, and the filtrate is measured with an automatic analyzer. The entire operation process including extraction and instrumental analysis was carried out in a normal air-conditioned environment at 20°C-30°C. the

3. Experimental analysis:

In order to investigate the effect of dialysis treatment on the detection results, the experiment carried out absorbance scanning on the extracts of tobacco samples after dialysis treatment and without dialysis treatment, and compared the absorbance spectra after chloride ion color development. the

① When dialysis treatment is not used, the background color of the tobacco sample extract will seriously interfere with the chloride ion detection results, and the shorter the measurement wavelength, the stronger the interference signal. The chromaticity diagram is shown in Figure 2. the

In Figure 2, the ordinate is the absorbance value, and the abscissa is the wavelength value; Standard2 is the chlorine ion standard solution curve after color development, and Sample1 is the sample extract without color development and without dialysis treatment. the

②Under the same measurement wavelength, the extracts of different varieties of tobacco samples have different strengths of interference signals; and the darker the color of the extract, the more serious the interference is. The results are shown in Table 1 and Figure 3. the

The absorbance (at 480nm) of table 1 sample extract

In Fig. 3, the ordinate is the value of the peak height of each sample, and the abscissa is the peak time, in seconds. the

③The dialysis treatment can effectively eliminate the interference of the background color of the tobacco sample extract, as shown in Figure 4 for the chromaticity diagram. the

In Fig. 4, the ordinate is the absorbance value, and the abscissa is the wavelength value; Sample1 is the sample extract curve without dialysis treatment, and Sample4 is the sample extract solution curve after dialysis treatment. the

Experimental conclusion: the background color of the tobacco sample extract will interfere with the detection results of chloride ions by the continuous flow method, and dialysis treatment can effectively eliminate this interference factor. Therefore, although the current domestic standard (YC/T162-2002, "Tobacco And the continuous flow method for the determination of chlorine in tobacco products "[S]) does not recommend the use of dialysis membranes, but it is adopted in the present invention. the

The selection experiment of embodiment 2 detection wavelength

The processing and analysis of instruments, samples, main reagents and samples are the same as in Example 1. the

Experimental analysis: On the basis of determining the need to use dialysis membrane to pre-treat the tobacco sample extract, through further research on the absorbance spectrum and the analysis of the test results of the standard tobacco leaf samples at each measurement wavelength, to determine the chlorine content of the tobacco sample. Appropriate wavelength for content detection. the

① It can be seen from Figure 2 that the maximum absorbance after chlorine ion color development is near the wavelength of 460nm. In the range of 450nm-490nm, the change of absorbance with the wavelength is small, and the influence of 5nm adjustment on the absorbance of the sample is less than 3%. Absorbance change ΔA/2 > 3% at 5 nm. That is to say, the photometric wavelength range of 450nm-490nm, excluding the endpoint values of 450nm and 490nm, is the characteristic absorption band for color development of chloride ions. the

②Amplify the absorbance spectrum of the dialysis-treated tobacco sample extract, as shown in Figure 5. It can be seen that when the measured wavelength is ≥489nm, the background color of the sample extract basically does not interfere with the absorbance value. When the measurement wavelength is reduced to 489nm-480nm, a weak interference absorbance value begins to appear; when the detection wavelength is less than or equal to 480nm, the increase in the interference absorbance begins to climb, and becomes larger; it tends to ease again near 400nm, and reaches the maximum interference absorbance value. Judging from this, for the tobacco sample extract, even after dialysis treatment, there is still a large interference photometric absorption at a wavelength of ≤480nm. Therefore, the appropriate photometric wavelength range for the detection of chloride ions in tobacco samples should be 480nm-490nm, excluding the two endpoints of 480nm and 490nm. the

In Fig. 5, the ordinate is the absorbance value, the abscissa is the wavelength value, and the wavelength value in the box of the abscissa is 488.99nm. Curve Sample4 is the sample extract curve that has been dialyzed but has not yet developed color,

③The detection results of the standard tobacco leaf samples are shown in Table 2. It can be seen that when the measurement wavelengths are 481nm, 485nm, 489nm and 490nm, the detection results of each sample are within the calibration range. At the rest of the measurement wavelengths, the test results of some rounds exceeded the calibration value, especially for samples with high chlorine content, and the deviation was obvious. the

The measured value (unit: %) of tobacco standard sample under the different measuring wavelengths of table 2

Experimental conclusion: Although 460nm is the maximum absorption wavelength of chloride ions in the thiocyanate chromogenic method, it is also the recommended detection wavelength for most standards such as GB13580.9-92 and HJ/T27-1999, but for tobacco samples at this luminosity There is strong interference near the wavelength, even at the 480nm wavelength originally used in the tobacco testing standard (YC/T162-2002), there will still be some interference, so it is not suitable for use. Comprehensive ①②③point analysis can judge that 481nm to 489nm, including 481nm and 489nm, is the suitable measurement wavelength for carrying out the continuous flow analysis method of chlorine content in tobacco samples and selecting 485nm is the best choice of the present invention. the

The experiment of embodiment 3 color reaction time

The processing and analysis of instruments, samples, main reagents and samples are the same as in Example 1. the

Experimental analysis: We adjusted the color reaction time of the sample by adding spiral tubes. The chromaticity diagram of the sample is shown in Figure 6. the

① The difference in reaction time will affect the absorbance of the sample color development, and the absorbance value is the largest under the condition of 664 seconds, that is, the condition of 10 turns of the spiral tube. the

② After the color reaction time was increased to 660 seconds, the change of absorbance was small with the extension of reaction time, and the absorbance value remained basically unchanged near the detection wavelength of 485nm. the

The curves in Figure 6 are the chromaticity diagrams of Sample3-10 turns spiral tube, Sample5-20 turns spiral tube, Sample4-0 turns spiral tube, Sample1-5 turns spiral tube from top to bottom, and the color development time is 664 seconds respectively , 701 seconds, 630 seconds, 648 seconds. the

Experimental conclusion: Ensuring the color reaction time of 660-670 seconds can ensure the stability of the detection results and obtain a higher instrument response value. This is consistent with the conditions of most chromogenic assays of chlorine content and is not greatly affected by the adjustment of the detection wavelength. the

The selection experiment of embodiment 4 colorimetric cell

Instrument, sample and main reagent and the processing and analysis of sample are the same as embodiment 1

Experimental analysis: By comparing the size of the response signals of different optical path colorimetric cells, and comparing the detection results affected by the detection period when different types of colorimetric cells are used, the effect of the colorimetric cell can be understood. the

In Fig. 7, the ordinate is the peak height value, and the abscissa is time, and the unit is second. From top to bottom are the light absorption curves of 50mm, 30mm, and 10mm colorimetric cells. the

① It can be seen from Figure 7 that with the increase of the optical path length of the cuvette, the response value of the instrument will increase accordingly, and the ratio of the response value is close to the ratio of the optical path length. At the same time, the greater the optical path length of the colorimetric cell, the more obvious the influence of the solute diffusion effect on the instrument—the response peak of the 50mm colorimetric cell is the sharpest, and the lack of leveling process affects the accuracy of the test results; while the 10mm colorimetric cell There is a long period of flat peak in the color cell, indicating that the response value of the instrument has been stabilized, thus effectively guaranteeing the accuracy and stability of the test results; the 30mm color cell is between the first two, and the detection value of the instrument has basically stabilized . the

② From the situation in Table 3, it can be judged that for the 30mm colorimetric cell, the appropriate sample detection time cycle setting should be the sampling time and injection interval time of 80s+80s; for the 10mm colorimetric cell, the appropriate setting is 70s +70s; while the instrument response value of the 10mm non-defoaming colorimetric cell is similar to that of the ordinary 10mm colorimetric cell, but it is much less affected by the solute diffusion effect, so its detection time period is also the shortest, and its setting is 60s+30s. the

Table 3 Influence of colorimetric pool

Remarks: 1. It means that there is no stable stage at the top of the signal peak, and the reaction has not reached static equilibrium. ○ indicates that the reaction has reached a static equilibrium. the

2. ◆ indicates that the signal peak is tailed, and the test result of the latter sample is interfered by the former sample. ◇ means no interference. the

Experimental results:

①The use of 10mm non-foaming colorimetric cell can effectively reduce the influence of solute diffusion effect and greatly improve the efficiency of instrument detection. the

② The instrument response value of the 10mm non-foaming cell is very low, so it is necessary to try to improve the signal response of the instrument, that is, to select the dialysis solvent and adjust the dilution ratio. the

Embodiment 5 Dialysis solvent selection experiment

Instrument, sample and main reagent and the processing and analysis of sample are the same as embodiment 1

Experimental analysis: The solvents used in the chlorine content detection are: water - the solvent of all reagents, 5% acetic acid solution - the sample extract and the rinse solution of the instrument, 1M nitric acid solution - one of the color reagents Solvent, methanol solution - Another solvent for the developer. Because the use of methanol solution will cause bubbles in the pipeline, which is not conducive to the detection, so it is not considered in this experiment. In the experiment, water was used as the main solvent first, and the dialysis results of different solutions were checked; after the dialysis solvent was determined, the situation when different solvents were used as the main solvent were tried. the

Table 4 Analysis of Dialysis Solution

Note: For each group of experiments, the average value of the three test results is used as the reporting data. the

Data that do not meet the relevant requirements are highlighted in bold. the

Table 5 Analysis of mainstream solution

Note: For each group of experiments, the average value of the three test results is used as the reporting data. the

Data that do not meet the relevant requirements are highlighted in bold. the

① From the data in Table 4, it can be judged that when water is selected as the dialysis solvent, the response value of the instrument is obviously low; when 5% acetic acid solution is selected, the fluctuation of the detection results is relatively large; only when nitric acid solution is used as the dialysis solvent, the instrument’s response value The peak value of the response is the largest, and the detection result is also very stable. the

② Judging from the data in Table 5, when 5% acetic acid solution is selected as the main solvent, the instrument response value is obviously low; while water has the highest instrument response value, and it is also the simplest solvent. the

Experimental conclusion: for the purpose of obtaining the highest sensitivity of the instrument and ensuring a certain stability, under this condition, when 1M nitric acid solution is used as the dialysis solvent and water is used as the main solvent, the effect of dialysis treatment is the best, and the response value of the instrument is Highest. the

Embodiment 6 Dilution ratio adjustment experiment

Instrument, sample and main reagent and the processing and analysis of sample are the same as embodiment 1

Experimental analysis: In order to ensure that the color reaction time remains unchanged throughout the detection process, the total flow rate of the solvent (=color solvent flow+main solvent flow) is controlled to be 2.20 ml/min in the test. The test adjusts the dilution ratio of the sample by adjusting the injection volume and the flow rate of the dialysis solvent, and then compares the correlation coefficient values obtained by the standard curve. Among them, the concentration of the standard solution covers the range of 0.142%-1.424%, which effectively covers the possible chlorine content of the tobacco leaf sample. Ion content, peak response with the sample, using a standard solution with a chloride ion content of 0.641% as a sample, and analyzing the spectrogram of the standard curve. The experimental results are shown in Table 6. the

Table 6 Analysis of Flow Design

Note: Bold fonts indicate values that meet the target criteria. the

According to the analysis in Table 6, the following conclusions can be drawn:

①Only within a certain sample dilution ratio range, the correlation coefficient value of the standard curve can reach above 0.999. the

②Increasing the amount of color-developing solvent (the color-developing solvent used in the test is already a saturated solution, so this purpose can only be achieved by increasing the flow rate), can effectively improve the response peak of the sample (peak height>500bit), and make the relatively When the dilution ratio is low, the curve will not be parabolic, so the correlation coefficient value can reach above 0.999. the

Experimental conclusion: Under the conditions of chromogenic solvent flow rate of 1.20ml/s, main solvent flow rate of 1.00CC/M, dialysis solvent flow rate of 0.80CC/M, and sample injection volume of 0.32CC/M, the analyzer can obtain a relatively high response value. And for the standard curve that effectively covers the chlorine content range of tobacco samples, the correlation coefficient value can reach more than 0.999. the

Embodiment 7 The present invention method measurement result accuracy verification experiment

Experimental analysis: the recovery rate is measured by the standard addition method, that is, samples with known content are used. In this embodiment, standard tobacco powder samples GBW08514 and GBW08515 are used, and a certain volume of standard solution containing chloride ions is added, and then detected. The detection value was compared with the calculated value to obtain the spike recovery of the method. Detected according to the assay method of the present invention, the specific workflow can refer to Figure 8 to obtain the detected value, and the calculated recovery results are shown in Table 7. the

Table 7 Recovery rate test

Note: 3 rounds of detection were carried out for each group of experiments, and the median was used as the reported data. the

Compare the accuracy of the results of the same sample, in different units, and with different methods to verify the accuracy of the detection scheme. The experimental results are shown in Tables 8 and 9. the

Table 8 Comparative test

Notes: 1. The detection methods adopted by Qingzhou Tobacco Research Institute are ion chromatography, spectrophotometry and Mohr method (silver nitrate volumetric method). the

2. The new detection method in Guangzhou Analysis and Test is automatic potentiometric titration. the

3. For analyzer detection, the average value of three measurement results is used as the reporting data. the

Table 9 paired tests

Notes: 1. For analyzer detection, the average value of three measurement results is used as the reported data. the

2. The new detection method in Guangzhou Analysis and Test is automatic potentiometric titration. the

① The recovery rate of the detection scheme of the present invention is in the range of 101.7%-104.6%, indicating that the measurement results of this scheme are relatively accurate. the

②The data obtained by testing the samples according to this scheme is very close to the results obtained by different testing methods by the national authorized quality inspection agency, which shows that the testing data of this scheme is more credible and has high accuracy. the

② Hypothesis testing with paired experiments ( $t={\mathrm{no}}^{1/2}\&Center\; Dot;\stackrel{\&OverBar;}{d}/S_d$ , d ₁ = _xi -y ₁ ) Further analysis of the test data shows that the t value is 1.6278, which is smaller than t _1-a/2 (n-1)=1.7341 (a=0.10, n=19). Even at the significance level of a=0.10, the detection method of the present invention has little difference with the classic automatic potentiometric titration method in detection results.

Experimental conclusion: the determination method of the chlorine content in tobacco of the present invention is compared with the existing continuous flow analysis method, and the optimal conditions for ensuring the accuracy of the results of the determination of the chlorine content in tobacco by the continuous flow analysis method have been determined. , On the basis of the measurement wavelength of 485nm±4nm, the color reaction time of the sample is determined to be around 670 seconds, and a 10mm non-foaming colorimetric cell is used, and a suitable dialysis solvent and dilution ratio are set, so that the detection cycle is changed from the original 160 seconds is shortened to 90 seconds, which greatly improves the efficiency; the detection results of the method of the present invention are closer to other detection schemes such as classical detection methods, ion chromatography, etc., and meet the requirements of accurate, stable, fast and high reliability of data. the

Claims (2)

1. the assay method of a Chlorine in Tobacco, discharge thiocyanate radical by chlorine and mercuric thiocyanate reaction, and then with developer in ferric iron complexing colour developing, use spectrophotometry again, adopt the detection wavelength of dialysis treatment, 37 ℃ of colour developings, OK range during spectrophotometry and select 660～670 seconds chromogenic reaction time, it is characterized in that:

Adopt 485nm ± 4nm to detect wavelength;

The dialysis solvent adopts the 1M salpeter solution, and water is as main solvent;

The flow velocity proportioning of developer and main solvent is 1.20 ml/min: 1.00 ml/min;

The diluted sample ratio is that sample size and dialysis solvent flow rate proportioning are 0.32 ml/min: 0.80 ml/min;

Colorimetric pool is not de-bubble of a 10mm colorimetric pool.

2. the assay method of Chlorine in Tobacco according to claim 1 is characterized in that described detection wavelength is 485nm.

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