Method for rapidly analyzing glucose in tobacco leaves based on reflection spectrum

Abstract

The invention discloses a method for rapidly analyzing tobacco leaf glucose based on a reflection spectrum, which comprises the following steps: S1, extraction: taking a flue-cured tobacco sample in a centrifuge tube, adding ultrapure water, carrying out vortex oscillation at room temperature, standing, taking extract liquor, centrifuging, passing through a water-based filter membrane to obtain extract liquor, and diluting the extract liquor to obtain a to-be-analyzed solution; s2, analysis: selecting a glucose determination method by the test strip-portable reflectometer, after the determination is started, immersing two reaction areas of the glucose test strip into a to-be-analyzed solution, inserting the glucose test strip into an instrument adapter of the reflectometer in the later period of the reaction, enabling the reaction areas to face an instrument display screen, and reading a measured value of glucose concentration from the instrument display screen; s3, calculating: calculating the percentage content of glucose in the flue-cured tobacco according to a formula. The method combines the advantages of the test strip and the portable reflectometer, realizes rapid and accurate determination of the glucose content in the tobacco leaves, and has the advantages of simple operation, low cost and convenient on-site detection compared with the traditional method.

Description

Technical Field

[0001] The invention relates to a method for quickly analyzing tobacco leaf glucose based on reflection spectroscopy, belonging to the technical field of tobacco leaf glucose analysis. Background Art

[0002] Tobacco contains a large amount of water-soluble sugars, including monosaccharides, disaccharides and other soluble oligosaccharides, such as glucose, fructose, maltose, sucrose, etc. These water-soluble sugars can be thermally decomposed under combustion conditions to produce new compounds such as furans, thereby affecting the aroma and taste of cigarettes, and the effects of different types of sugars vary. Literature reports that glucose can enrich various types of volatile components in smoke, especially aldehydes, ketones and nitrogen-containing heterocyclic aroma substances, while fructose has little effect on smoke volatiles. Water-soluble sugars are also precursors of important latent aroma substances. During the baking process, glucose can react with amino acids through the Maillard reaction to form a large number of Amadori compounds. At the same time, water-soluble sugars will also affect the physical appearance, such as tobacco leaves with a high content of soft, elastic and bright color. Glucose is an important chemical component in tobacco, generally accounting for 5%-15%, and is one of the important factors affecting the quality of tobacco products. Glucose in cigarettes can enrich the aroma of cigarettes, improve the roundness of smoke, and give tobacco products a unique flavor. Within a certain range, the higher the glucose content in tobacco leaves, the better the quality of the tobacco leaves. However, too high a glucose content will also make the smoke taste bland, weaken its strength and intensity, deteriorate its combustibility, and increase its tar. Therefore, it is of great significance to quickly and accurately determine the glucose content in tobacco.

[0003] Glucose determination is an important part of tobacco chemical analysis. Currently, the commonly used determination methods mainly include gas chromatography, high performance liquid chromatography and ion chromatography. Gas chromatography is a highly sensitive and fast analysis method, but for glucose determination, derivatization is required for analysis, and the operation is relatively complicated, which limits the practical application of this type of method; high performance liquid chromatography-differential refractive index detection is a fast and direct sugar analysis method, but it is sensitive to temperature and has low sensitivity; while evaporative light scattering detection has good stability and higher sensitivity than differential refractive index, which is suitable for the analysis of low-content sugar substances, but solid phase extraction columns are also required to purify impurities; ion chromatography has the advantages of high sensitivity, no derivatization, easy operation and no use of toxic chemical reagents, but the sample analysis time is long and the analysis efficiency is relatively low. It can be seen that the glucose analysis method has shortcomings in ease of use, portability and environmental friendliness. Therefore, it is necessary to develop a simple and accurate method to measure the glucose content in flue-cured tobacco and provide scientific data reference for tobacco leaf quality monitoring.

[0004] Test strips - portable reflectometer Reflectoquant analysis system has been widely used in on-site analysis and detection of samples such as plants, food and beverages, soil, and water bodies. It can be used to detect the content of nitrate, phosphate, potassium, calcium, ascorbic acid, hydroxymethyl furfural, etc. It has the advantages of small size, easy to carry, good stability, simple and fast operation, high sensitivity, and can be used for field operations. In addition, the reflectometer has a storage function, which can import the measured data into the computer for processing, so that the efficiency is further improved. The test strip method is a quantitative analysis method based on chemical reactions. The method has simple operation steps and is easy to use. It does not require professional operation for testing, and the test results can be obtained quickly. In addition, the test strip does not contain corrosive chemical reagents, which will not threaten human safety and health, and will not pollute the ecology and environment. The working principle of the glucose test strip is to oxidize glucose to gluconolactone under the catalysis of glucose oxidase. At the same time, the hydrogen peroxide formed in the process reacts with the organic redox indicator to form a blue-green dye under the action of peroxidase, so that the glucose content in the sample can be determined by the reflectometer. This method is mainly used for the quantitative analysis of glucose in simple matrices such as juice and honey. Studies have reported that the flue-cured tobacco matrix is ​​complex, with high levels of interfering substances such as terpenes, alkaloids, flavonoids and coumarins. There are also structural analogs that interfere with the catalytic reaction of peroxidase, such as fructose, inositol, sucrose, etc. In addition, the pigments in tobacco leaves will also affect the color of the test strips and interfere with the intensity of reflected light. Therefore, the above factors will lead to a strong matrix effect during the detection process, resulting in a large error in the quantitative results. It is reported that the matrix effect causes the test results to deviate by 50%. Summary of the invention

[0005] Based on the above, the present invention provides a portable, easy-to-operate, rapid, accurate and low-cost method for analyzing glucose in tobacco leaves to overcome the shortcomings of the prior art.

[0006] The technical solution of the present invention is: a method for quickly analyzing glucose in tobacco leaves based on reflectance spectroscopy, comprising:

[0007] S1 Extraction: Take the flue-cured tobacco sample in a centrifuge tube, add the extraction solvent, vortex and oscillate at room temperature for a certain period of time, let it stand, centrifuge the extract, pass it through a water filter membrane to obtain the extract, and dilute the extract to obtain the solution to be analyzed;

[0008] S2 Analysis: Test Strip - Portable Reflectometer Select the glucose determination method. After starting the determination, immerse the two reaction areas of the glucose test strip into the liquid to be analyzed. In the later stage of the reaction, insert the glucose test strip into the instrument adapter of the reflectometer, with the reaction area facing the instrument display screen, and read the measured value of glucose concentration from the instrument display screen;

[0009] S3 calculation: Calculate the percentage of glucose in flue-cured tobacco according to the following formula:

[0010] w(%)=C×D×V×10 -3 ×100% / m;

[0011] Where, C is the concentration of glucose measured by reflectometer (mg / mL), D is the dilution factor, V is the volume of the extract (mL), and m is the sample weight (mg).

[0012] Preferably, in step S1, the extraction solvent is 10-30 mL of ultrapure water, and the vortexing is performed alternately by vortexing and standing, each for 5 minutes, repeated 3 times, and the total extraction time is 30 minutes.

[0013] Preferably, in step S2, the reflectometer is set to 60s timing when the measurement starts, the two reaction areas of the glucose test strip are immersed in the liquid to be analyzed for 15-30s at a temperature of 15-30°C, and the test strip is inserted into the instrument adapter of the reflectometer in the remaining 10s.

[0014] Beneficial effects of the present invention: The present invention is based on vortex oscillation extraction of aqueous solution, test strips combined with portable reflectometers, to construct a simple, rapid and accurate quantitative analysis method for glucose in flue-cured tobacco. The method does not require purification of the extracted sample, the analysis is rapid, the environmental conditions have little effect on the determination, the accuracy is high, and the matrix effect is low under the complex matrix conditions of tobacco leaves. At the same time, due to the short detection time, the error caused by the hydrolysis of disaccharides such as sucrose is reduced.

[0015] The present invention optimizes extraction and reflectometer conditions, analyzes matrix effects, and evaluates interfering substances. Under the best analytical conditions, the glucose mass concentration is linear in the range of 1-100 mg / L, the regression equation is y=0.9952x+0.0253, and the correlation coefficient is 0.9991; the matrix effect is manifested as matrix weakening and the quantitative relative error caused by the matrix effect is less than 2.5%; the extraction recovery rate of the method is 95.4-98.5%, and the repeatability and reproducibility are 1.39-2.07% and 2.25-3.54% respectively. At the same time, the main metabolites in tobacco, fructose, sucrose, inositol, maltose, nicotine, etc., will not interfere with the determination of glucose. The oximation-silylation-gas chromatography-mass spectrometry analysis method is compared with the present method, and the quantitative determination result is less than 5%, and the method meets the verification requirements of the International Council for Harmonization (ICH) guidelines. In summary, the present invention combines the advantages of the test strip and the portable reflectometer to achieve rapid and accurate determination of the glucose content in tobacco leaves. Compared with traditional methods, the method of the present invention has the advantages of simple operation, low cost and convenience for on-site detection, and provides an effective analysis technology for tobacco leaf quality monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1It is the linear correlation analysis of the test results of the test strip-reflectometer method and the silanization derivatization method;

[0017] Figure 2 Standard curve analysis for different concentrations of glucose;

[0018] Figure 3 In the figure, A is a schematic diagram of the test strip and the reflectometer, B is a schematic diagram of the standard sample detection, C is a schematic diagram of the gradient standard sample detection, and D is a schematic diagram of the sample detection. DETAILED DESCRIPTION

[0019] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0020] 1. Instruments and reagents

[0021] Test Strips - Portable Reflectometer Selection 20 reflectometer (Merck, Germany), glucose test strips (Chromatography Department), gas chromatography-mass spectrometry 7890A-5975C equipped with CTC PAL autosampler (Agilent, USA), and its pre-treatment instruments include nitrogen blower, ultrasonic cleaning device, constant temperature metal bath, multi-tube vortex mixer and centrifuge. Glucose standard (purity, >99.5%), internal standard phenyl-β-D-pyranoglucopyranoside hydrate (TCI, ≥99%). Derivatization reagents methoxyamine hydrochloride (MEOX, ≥98%), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, ≥98%), and anhydrous pyridine (≥99.5%) were purchased from Sigma. The system obtained ultrapure water for extraction, typical flue-cured tobacco samples were used for method optimization, and 20 tobacco leaf samples with a wide range of reducing sugar distribution were screened for method comparison.

[0022] 2. Preparation of standard solution

[0023] Accurately weigh 501.3 mg of glucose into a 10 mL volumetric flask, dissolve it in ultrapure water and make up to volume to prepare a 50.13 mg / mL standard stock solution. Dilute the standard stock solution to prepare 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L, and 90.0 mg / L standard working solutions. Prepare a 10.25 mg / mL standard working solution of phenyl-β-D-pyranoglucoside with acetonitrile / water (v / v 1:1).

[0024] Embodiment 1:

[0025] A method for rapid analysis of glucose in tobacco leaves based on reflectance spectroscopy, comprising:

[0026] S1 extraction: accurately weigh 200 mg of flue-cured tobacco sample into a 50 mL centrifuge tube, add 20 mL of ultrapure water, vortex and oscillate at 2500 rpm for 5 min at room temperature, let stand for 5 min, repeat extraction 3 times for a total of 30 min, take 1.5 mL of the extract and centrifuge at 12000 rpm for 5 min, filter through a water filter to obtain the extract, take 100 μL of the extract and put it into a 2 mL centrifuge tube, dilute to 2 mL and mix well to obtain the solution to be analyzed;

[0027] S2 Analysis: Preheat the test strip-portable reflectometer for more than 30 minutes, and use the calibration barcode and blank test strips to calibrate the instrument (clean and recalibrate the reflectometer every 100 samples. Use solvent matching calibration curve to obtain the glucose concentration of the extract). Select the glucose determination method (1-100 mg / L), and press the START button of the reflectometer to start the 60s timing measurement of the reflectometer. At the same time, immerse the two reaction areas of the glucose test strip in the sample solution for 15-30 seconds (15-30°C), ensuring that both reaction areas are immersed, shake off the excess liquid and use lens paper to absorb the non-reaction area solution. When there are 10 seconds left, insert the test strip into the instrument adapter of the reflectometer, with the reaction area facing the instrument display. After the countdown ends, the LCD displays the result, that is, the measured value of the glucose concentration is displayed in mg / L.

[0028] S3 calculation: Calculate the percentage of glucose in flue-cured tobacco according to the following formula:

[0029] w(%)=C×D×V×10 -3 ×100% / m;

[0030] Wherein, C is the concentration of glucose measured by reflectometer (mg / mL), D is the dilution factor, V is the volume of the extract (mL), and m is the sample weight (mg). In this embodiment, D, V and m are 20, 20 mL and 200 mg, respectively.

[0031] Comparative Example 1:

[0032] The method for analyzing glucose in tobacco leaves based on gas chromatography-mass spectrometry comprises:

[0033] S1 extraction: accurately weigh 200 mg of flue-cured tobacco sample into a 50 mL centrifuge tube, add 200 μL of 10.25 mg / mL phenyl-β-D-pyranoglucoside solution, add 20 mL of ultrapure water, vortex at 2500 rpm for 5 min at room temperature, let stand for 5 min, repeat extraction 3 times for a total of 30 min, take 1.5 mL of the extract and centrifuge at 12000 rpm for 5 min, filter through a water filter to obtain the extract, take 20 μL of it and place it in a 2 mL chromatographic bottle for glucose derivatization;

[0034] S2 derivatization: 20 μL of the extraction solution was dried with nitrogen, 300 μL of methoxyamine hydrochloride solution (pyridine) was added, and the mixture was incubated at 80°C in a water bath for 30 min, and cooled at room temperature. Then 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide was added, and the mixture was incubated at 80°C in a water bath for 60 min, and allowed to stand at room temperature for 30 min. The reaction solution was directly analyzed by gas chromatography-mass spectrometry;

[0035] S3 analysis: Gas chromatography-mass spectrometry instrument conditions: GC-MS was used for qualitative and quantitative analysis of glucose. The injection port temperature was 280°C, the injection was in split mode, and the split ratio was 10:1. Ultrapure helium was used as the carrier gas, and the flow rate was 1.0 mL min in constant flow mode. -1 HP-5MS column (60m×0.25mm inner diameter×0.25μm film thickness) was used for separation. The column oven temperature was programmed as follows: 60℃ for 2min, 5℃min -1 Increase to 230℃, hold for 5min, then 8℃min -1 Increase to 290℃, maintain for 11.5min, and run time for 60min. The transfer line temperature and solvent delay were 280℃ and 12.0min, respectively. The temperatures of the ion source and quadrupole were 230℃ and 150℃, respectively. All mass spectral data were obtained at 70eV of electron impact ionization source (EI). The mass-to-charge ratio (m / z) range of full scan mass spectrometry acquisition was 45-600amu. The derivative products of glucose and phenyl-β-D-pyranoglucoside were MEOX-5TMS (36.998min and 37.437min) and 4TMS (46.551min), respectively. Selected ion scanning quantification, the quantitative ion of glucose derivative product was m / z 319, and the qualifier ions were m / z 205 and 160; the quantitative ion of internal standard phenyl-β-D-pyranoglucoside derivative product was m / z 361, and the qualifier ions were m / z 271 and 243.

[0036] 1. Accuracy analysis

[0037] 20 tobacco leaf samples with a wide range of reducing sugar distribution were selected, and the glucose content was determined and compared using Example 1 and Comparative Example 1 (silylation derivatization-gas chromatography-mass spectrometry). Figure 1The linear relationship between the two methods was shown. The two methods had a significant linear correlation (P < 0.01), and the Pearson coefficient was 1.000. The linear regression equation was y = 1.021x + 0.645, R 2 =0.999, which basically conforms to the relationship of 1:1, proving the accuracy of the method and comparable to the classical method. At the same time, since the test strip method is a matrix weakening effect, while the literature reports that the silanization derivatization method is a matrix enhancement effect, the slope of the equation is greater than 1, the overall measurement value is smaller, the slope error is 2.1%, and the measurement error is less than 5%. However, due to the small difference in the measurement results, the results do not affect the evaluation of tobacco leaf quality. Studies have reported the comparison of the analysis of nitrate and 5-hydroxymethylfurfural content by test strip-portable reflectometer, ion chromatography and high performance liquid chromatography. The results showed that the correlation coefficient of the two methods for nitrate determination was 0.996, and the test strip-portable reflectometer was more accurate. At the same time, the test strip-portable reflectometer also has the characteristics of high ease of use, low cost and good portability, and can be a method for rapid on-site detection. Due to the short detection time, the error caused by the hydrolysis of disaccharides such as sucrose is reduced, further improving the accuracy of the method.

[0038] 2. Matrix effect and metabolite interference analysis

[0039] In order to evaluate the matrix effect of glucose in flue-cured tobacco in the detection of the present invention, an interference factor (f) is introduced for evaluation. The interference factor (f) is the quotient of the detection concentration (C) of glucose in the matrix and the actual concentration (RC). By adding a standard sample of known concentration (low 20 mg / L and high concentration 40 mg / L) to the matrix, the interference factor of glucose in the tobacco matrix can be obtained.

[0040] f=(C 加 -C) / RC

[0041] C 加 ——Detection concentration after addition; C——Detection concentration without addition; RC——Actual concentration after addition

[0042] According to the definition of interference factor, when the interference factor (f)>1, the matrix effect shows an enhancement effect; when (f)=1, there is no matrix interference effect; when (f)<1, the matrix effect shows a weakening effect. The results are shown in Table 1. When glucose in tobacco leaves was determined by test strips and portable reflectometer method, the f of glucose was less than 1, showing a matrix weakening effect, and the quantitative relative error caused by the matrix effect was less than 2.5%, indicating that the matrix effect was not significantly weakened and would not have a significant impact on the glucose quantification.

[0043] Table 1 Analysis of matrix interference factors of glucose

[0044]

[0045] Further addition of fructose, sucrose, inositol, malic acid and nicotine, metabolites with high content in tobacco, was performed to determine the effects of blank and metabolite addition on the determination results to evaluate the matrix effect of the metabolite matrix. The results in Table 2 show that the average value of glucose determination was 49.4-51.5 mg / mL in low, medium and high concentrations of metabolite addition, and the metabolite matrix had no significant effect on glucose quantification, further proving that the method has good stability and low matrix effect. In summary, the method of determining glucose in tobacco leaves by test strips and portable reflectometer has low matrix effect, no matrix correction method is required, good stability, and is suitable for rapid determination of glucose content in tobacco leaves.

[0046] Table 2 Effects of different metabolite matrices on the determination results

[0047]

[0048] 3. Method validation analysis

[0049] The glucose test strip method was validated using linearity (external standard calibration curve), sensitivity (measurement range), accuracy (extraction recovery), and precision (repeatability and reproducibility). A solvent matching-calibration curve was constructed using a weighted (1 / X) least squares linear regression model over the range of 1-100 g / L. The linear curve is shown in Figure 2. Figure 2 As shown, y=0.9952x+0.0253, and the correlation coefficient is 0.9991. According to the weight of the sample, the dilution ratio and the recovery rate, the measurement range of glucose in flue-cured tobacco is about 0.21%-20.58%.

[0050] Table 3 Recovery, repeatability and reproducibility of glucose in flue-cured tobacco by reflectometer analysis method

[0051]

[0052] Accuracy was assessed by adding low or high levels of glucose to the sample and measuring the recovery (%) six times. Precision was assessed using repeatability and reproducibility, where repeatability (intra-day precision) and reproducibility (inter-day precision) were calculated by analyzing five independent samples within 1 day and 3 days, respectively. Table 3 shows that the average recovery of potassium in flue-cured tobacco was between 95.4% and 98.5%. Repeatability and reproducibility were 1.39% to 2.07% and 2.25% to 3.54%, respectively. The established water extraction, test strip combined with reflectometer detection method has high accuracy and precision, and meets the validation requirements of the International Council for Harmonization (ICH) guidelines.

[0053] Below is the optimization test of the parameters of the method of the present invention:

[0054] 1. Optimization of glucose extraction conditions

[0055] Water-soluble sugars in tobacco are generally extracted using water, acetonitrile, ethanol-water solution and dilute sodium hydroxide solution. Since the test strip is coated with a stationary phase and the optimal pH for detection is 2-10, the organic solvent in the extraction solution will dissolve a large amount of the stationary phase and have a greater impact. In addition, tobacco contains a variety of acids such as citric acid and malic acid. The water extract of tobacco samples is also weakly acidic (pH 4-6 range), which makes it within the optimal range and does not require extraction with dilute sodium hydroxide solution. Therefore, in order to achieve the requirements of green chemical extraction, water is selected as the extraction solvent. Studies have reported that long-term storage (>2h) of weakly acidic water extraction solutions will lead to the hydrolysis of sucrose, but the test strip method has a fast detection speed and does not require sample waiting. The low sucrose hydrolysis rate will not affect the quantification of glucose. The water extraction solvent was optimized under different extraction methods and times (Table 4). First, vortex oscillation, homogenization and ultrasonic extraction were selected. The results showed that the extraction efficiency of the three methods was similar, but the repeatability of vortex extraction was better. The possible reason was that vortex oscillation could fully mix the extraction solvent with tobacco powder and improve the wettability of the sample. The extraction time was further optimized. The results showed that the best extraction efficiency was stable at 30 minutes, which could better extract glucose from tobacco leaves.

[0056] Table 4 Comparison of different extraction methods and time

[0057]

[0058] 2. Optimization of reflectometer analysis conditions

[0059] The optimization of the reflectometer measurement conditions is mainly the time the test strip is immersed in the glucose solution. If the time is too short, the test strip cannot be completely immersed, resulting in a low measurement value; if the time is too long, a large amount of the coated stationary phase dissolves into the solution, resulting in a high measurement value. The immersion time was measured from 10, 15, 20, 30, 35, and 40 seconds, and the results showed that the measurement results of 15-30 seconds were accurate and stable, which is suitable for the determination of glucose in aqueous solution. Figure 3 AD are schematic diagrams of test strip and reflectometer, standard detection, gradient standard detection and sample detection, respectively.

[0060] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for rapidly analyzing glucose in tobacco leaves based on reflectance spectroscopy, characterized in that: include: S1 Extraction: Take the flue-cured tobacco sample in a centrifuge tube, add the extraction solvent, vortex and oscillate at room temperature for a certain period of time, let it stand, centrifuge the extract, pass it through a water filter membrane to obtain the extract, and dilute the extract to obtain the solution to be analyzed; S2 Analysis: Test strip - portable reflectometer Select the glucose determination method. After starting the determination, immerse the two reaction areas of the glucose test strip into the liquid to be analyzed. In the later stage of the reaction, insert the glucose test strip into the instrument adapter of the reflectometer, with the reaction area facing the instrument display screen, and read the measured value of glucose concentration from the instrument display screen; S3 Calculation: Calculate the percentage of glucose in the flue-cured tobacco according to the following formula: w(%)=C×D×V×10 -3 ×100% / m; Where, C is the concentration of glucose measured by reflectometer (mg / mL), D is the dilution factor, V is the volume of the extract (mL), and m is the sample weight (mg).

2. The method for rapid analysis of glucose in tobacco leaves based on reflectance spectroscopy according to claim 1, characterized in that: In step S1, the extraction solvent is 10-30 mL of ultrapure water, and the oscillation vortex extraction is performed by alternating oscillation vortex-standing for 5 minutes each, repeated 3 times, and the total extraction time is 30 minutes.

3. The method for rapid analysis of glucose in tobacco leaves based on reflectance spectroscopy according to claim 1, characterized in that: In step S2, the reflectometer is set to 60s timing at the beginning of the measurement, and the two reaction areas of the glucose test strip are immersed in the liquid to be analyzed for 15-30s at a temperature of 15-30°C. When the remaining 10s are left, the test strip is inserted into the instrument adapter of the reflectometer.

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