1 Introduction The water-soluble sugar in tobacco, especially reducing sugar is one of the most important chemical components that affect the taste of tobacco. At present, the measuring methods of reducing sugar and total sugar in tobacco mainly include redox titration, spectrophotometry, continuous Flow analysis and high performance liquid chromatography. Liquid chromatography is the most reliable method, but its sample preparation is more troublesome, and the inconsistency between the mobile phase and the extraction solvent will cause the refractive index detector to have a large solvent peak and interfere with the analysis. For the first time, we used WatersSugar-Pak1 calcium-type cation exchange column to separate and determine four sugars in tobacco samples, and established a sample pretreatment method of ultrasonic leaching with water and solid phase extraction preseparation, and obtained satisfactory results.
2 Experimental part
2.1 Main instruments and reagents STI 5000 high performance liquid chromatograph
2.2 Chromatographic conditions The mobile phase of the column is: 0.05 g / L EDTA
2.3 Sample processing Weigh accurately about 0.500 g of tobacco sample, ultrasonically extract with 50 mL of water at 40 ~ 50 ° C for 40 min, filter, discard the initial about 10 mL of filtrate, and collect the subsequent filtrate. Take 5 mL of the filtrate through a pre-activated Sep-PakC18 solid phase extraction cartridge, discard the first 2 mL, collect the next 3 mL and filter with a 4.5 μm filter membrane, and take 20 μL for injection analysis.
3 Results and discussion
3.1 Selection of experimental conditions The experiment uses a refractive index detector for detection. The calcium-type cation exchange column is a special column for separating sugar and polyhydroxy compounds. The column uses 0.05g / LEDTA calcium and sodium aqueous solution as the mobile phase. The column temperature is required to be controlled between 80 ~ 90 ℃, and the experiment uses 85 ℃.
3.2 Sample pretreatment Because the four sugars are easily soluble in water, the experiment uses water ultrasonic leaching method. The sugar dissolution is slow at room temperature, and the temperature can accelerate the sugar dissolution, but the temperature is too high (more than 60 ℃) will accelerate Polysaccharide, sucrose and maltose are hydrolyzed; therefore, the experiment chooses a leaching temperature of 40 ~ 50 ℃, at this temperature, ultrasonic leaching for more than 30min can completely dissolve the sugar, and the experiment uses ultrasonic leaching for 40min. The sugar extract is best measured on the same day. If left overnight, the glucose measurement result will be slightly higher. In addition to sugar, the tobacco sample extract contains a small amount of other impurities, which will contaminate the column and reduce the efficiency of the column, so these components should be pre-separated before injection. We use Sep-PakC18 solid phase extraction cartridge pre-solid phase extraction and Sep-Pak Alumin Cartridges guard column on-line separation can effectively remove impurities.
3.3 Regression equations, correlation coefficients and detection limits were prepared with standard solutions with concentrations of 5, 1, 0.2, 0.04, and 0.008g / L. After injection, the regression equation was calculated according to the peak areas of different concentrations. N = 3, the lowest detection concentration of each component was measured, the results were: sucrose: A (peak area) = 1431 + 1.954 × 108C (g / mL), r = 0.9998, detection limit was 1.5mg / L; maltose: A (peak area) = 997 + 1.921 × 108C (g / mL), r = 0.9998, detection limit is 1.5mg / L; glucose: A (peak area) = 1216 + 1.697 × 108C (g / mL), r = 0.9997, detection limit is 2.0mg / L; fructose: A (peak area) = 1349 + 1.943 × 108C (g / mL), r = 0.9999, detection limit is 2.0 mg / L.
4 Conclusion The calcium type cation exchange column high performance liquid chromatography is used to determine the sugar in the tobacco sample, and the sugar in the sample is extracted by ultrasonic leaching with water. The method is simple and fast, and the result is reliable. Requirements, improve work efficiency. The method was used to analyze several kinds of smoke samples, the recovery rate of each sugar was between 96% and 104%, and the relative standard deviation was between 0.98% and 1.36%. The result is satisfactory.
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