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单糖分离纯化总糖含量的测定的初步方式
来源:http://www.jnjrh.com 日期:2019-10-06 发布人:admin
采用苯酚-硫酸法停止糖含量测定,其原理是:糖在浓硫酸作用下,脱水生成的糠醛或羟甲基糠醛,再与苯酚缩合成一种橙红色化合物,在 200 ng~100 mg范围内其颜色深浅与糖的含量成正比,且在 490 nm波长下有大吸收峰,故可用比色法在此波长下测定。苯酚法可用于己糖、戊糖、甲基化的糖和多聚糖的测定,办法简单,灵活度高,实验时根本不受蛋白质存在的影响,并且产生的颜色160 min内稳定。
The content of sugar was stopped by phenol sulfuric acid method. The principle was that the sugar was dehydrated to form a furfural or hydroxymethyl furfural under the action of concentrated sulfuric acid. Then it was condensed with phenol to form an orange red compound. The color depth was proportional to sugar content in the range of 200 ng to 100 Mg, and the maximum absorption peak at 490 nm wavelength could be determined by colorimetric method at this wavelength. Phenol method can be used for the determination of hexose, pentose, methylated sugar and polysaccharides. The method is simple and flexible. It is not affected by the presence of protein at all in the experiment, and the color produced is stable within 160 minutes.
60%苯酚储藏液:称取60 g苯酚,溶解于40 mL蒸馏水中,4℃避光保管。用时加蒸馏水稀释至6%。
60% phenol storage solution: weigh 60 g phenol, dissolve in 40 mL distilled water, keep away from light at 4 C. Dilute to 6% with distilled water.
0.1 g/L糖规范溶液:精确称取葡萄糖10 mg,蒸馏水定容于 100 mL容量瓶中。
0.1 g/L sugar standard solution: accurately weigh 10 mg of glucose, distilled water must be contained in 100 mL capacity bottle.
规范曲线制造:移液管分别量取规范溶液0 mL、0.2 mL、0.4 mL、0.6 mL、0.8 mL、1.0 mL转于玻璃试管(1.8 × 18 cm)中,加蒸馏水补至1.0 mL,每个浓度反复三个样品,分别向每支试管中参加6%苯酚试剂 0.5 mL,浓硫酸2.5 mL,疾速振荡平均,室温放置30 min,在λ 490 nm处测定吸光值A。以A为纵坐标,糖含量C为横坐标,得规范曲线。
Manufacture of the standard curve: the pipette takes 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of the standard solution separately and transfers them to the glass test tube (1.8 *18 cm), adds distilled water to 1.0 mL, three samples of each concentration are added repeatedly, respectively, 6% phenol reagent is 0.5 mL, concentrated sulfuric acid is 2.5 mL, the speed of disease oscillates averagely, and is placed at room temperature for 30 minutes, and the absorbance value is determined at lambda 490 nm. A. With A as the ordinate and C as the abscissa, the standard curve was obtained.
样品中糖含量测定:取浓度0.1 g/L的样品溶液l mL,参加苯酚试剂0.5 mL,浓硫酸2.5 mL,按规范曲线同样操作,测定吸光值。按规范曲线计算总糖含量。
氨基磺酸
Sample sugar content determination: take 0.1 g/L sample solution 1 mL, participate in phenol reagent 0.5 mL, concentrated sulfuric acid 2.5 mL, according to the standard curve the same operation, determine the absorbance value. The total sugar content was calculated according to the standard curve.
酸性糖含量的测定
DETERMINATION OF ACID SUGAR CONTENT
采用间羟基联苯法测定酸性糖,即糖醛酸的含量。糖醛酸与浓硫酸在100 ℃条件下加热后,可与间羟基联苯发作颜色反响,在0.5~15 μg范围内其颜色深浅与糖醛酸的含量成正比,525 nm波长下有大吸收峰,故可用比色法在此波长下测定糖醛酸含量。本实验以D-半乳糖醛酸(GalA)为规范品。
The content of acid sugar, glucuronic acid, was determined by m-hydroxybenzene method. The color of glucuronic acid and concentrated sulfuric acid can react with m-hydroxybenzene when heated at 100 C. The color of glucuronic acid is directly proportional to the content of glucuronic acid in the range of 0.5-15 ug. There is a maximum absorption peak at 525 nm wavelength. Therefore, the content of glucuronic acid can be determined by colorimetry at this wavelength. D-galacturonic acid (GalA) was used as standard substance in this experiment.
0.5%氢氧化钠(w/v):称取0.1 g固体氢氧化钠,溶于20 mL蒸馏水中。
0.5% sodium hydroxide (w/v): 0.1 g solid sodium hydroxide is weighed and dissolved in 20 mL distilled water.
间羟基联苯试剂:称取30 mg间羟基联苯,溶于0.5%氢氧化钠水溶液中,定容至10 mL,4 ℃避光保管。
M-hydroxybenzene reagent: 30 mg m-hydroxybenzene was weighed, dissolved in 0.5% sodium hydroxide aqueous solution, constant volume to 10 mL, and stored at 4.
饱和氢氧化钾:称取5 g氢氧化钾,参加2 mL蒸馏水,充沛搅拌溶解,上部清液即为饱和氢氧化钾。
Saturated potassium hydroxide: weigh 5 g potassium hydroxide, take part in 2 mL distilled water, fully stir and dissolve, the supernatant is saturated potassium hydroxide.
氨基磺酸试剂:称取3.9 g氨基磺酸,加蒸馏水5 mL,滴加饱和氢氧化钾水溶液,充沛振荡至氨基磺酸完整溶解,冷却至室温,再继续滴加饱和氢氧化钾水溶液至pH 2.5,蒸馏水补充体积至10 mL,室温保管备用。
Amino sulfonic acid reagent: weigh 3.9 g amino sulfonic acid, add distilled water 5 mL, drop saturated potassium hydroxide solution, oscillate abundantly until amino sulfonic acid dissolves completely, cool to room temperature, then continue to drop saturated potassium hydroxide solution to pH 2.5, distilled water supplement volume to 10 mL, store at room temperature.
0.1 g/L D-GalA规范溶液:称取10 mg D-GalA,溶解于蒸馏水中,定容至100 mL。
0.1 g/L D-GalA standard solution: weigh 10 mg D-GalA, dissolve in distilled water, constant volume to 100 mL.
规范曲线制造:移液器分别量取0.1 g/L D-GalA规范溶液0 μL、50 μL、100 μL、200μL、300 μL、400 μL于玻璃试管(1.8 × 18 cm)中,加蒸馏水补至总体积400 μL,每个浓度做三个反复。分别向试管中参加40 μL氨基磺酸试剂,摇匀,再参加2.5 mL浓硫酸,振荡平均,沸水浴20 min。疾速冷却至室温后,向各管中参加间羟基联苯试剂40 μL,摇匀,室温放置15 min。在λ 525 nm处测定吸光值A。以A为纵坐标,D-GalA含量(μg)为横坐标,制造规范曲线。
Standard curve manufacturing: the pipette takes 0.1 g/L D-GalA standard solution of 0,50,100,200,300 and 400 mu L in glass test tube (1.8*18 cm) and adds distilled water to the total volume of 400 mu L. Each concentration is repeated three times. Participate 40 mu L aminosulfonic acid reagent in test tube, shake well, then participate in 2.5 mL concentrated sulfuric acid, shake averagely, boiling water bath for 20 minutes. After rapid cooling to room temperature, the m-hydroxybenzene reagent was added to each tube, shaken, and placed at room temperature for 15 minutes. Absorption A was measured at lambda 525 nm. With A as the ordinate and D-GalA content (ug) as the abscissa, the standard curve was manufactured.
样品中糖醛酸含量测定:取浓度0.1 g/L的样品溶液400 μL,按规范曲线制造办法,测定吸光值,依据规范曲线和样品浓度计算糖醛酸含量。
The determination of glucuronic acid content in the sample: Take the sample solution with 0.1 g/L concentration of 400 mL, according to the standard curve manufacturing method, determine the absorbance value, and calculate the content of glucuronic acid according to the standard curve and sample concentration.