WO2018040820A1 - 一种利用酸碱指示剂法测定壳寡糖脱乙酰度的方法 - Google Patents

一种利用酸碱指示剂法测定壳寡糖脱乙酰度的方法 Download PDF

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WO2018040820A1
WO2018040820A1 PCT/CN2017/095113 CN2017095113W WO2018040820A1 WO 2018040820 A1 WO2018040820 A1 WO 2018040820A1 CN 2017095113 W CN2017095113 W CN 2017095113W WO 2018040820 A1 WO2018040820 A1 WO 2018040820A1
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deacetylation
degree
chitosan oligosaccharide
acid
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苏政权
郭姣
蒋瑶
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广东药科大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G01N21/79—Photometric titration

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  • the invention belongs to the field of chemistry, and in particular relates to a method for determining the degree of deacetylation of chitooligosaccharides by using an acid-base indicator method.
  • Chitooligosaccharides are homopolymers or heteropolymers of glucosamine and N-acetylglucosamine linked by ⁇ -1,4 glycosidic bonds.
  • Chitosan oligosaccharide is a degradation product of chitosan, but compared with chitosan, chitosan oligosaccharide has the advantages of better solubility, lower viscosity and easier absorption by the human body. It has a wide range of fields in medicine, agriculture, fine chemicals and other fields. application. Chitosan oligosaccharide not only has anti-tumor, antibacterial, anti-oxidant and other functions, but also has biological functions such as weight loss, lipid regulation and immunity enhancement.
  • the degree of deacetylation of chitooligosaccharides can affect the biological, physical and chemical functions and activities of chitosan oligosaccharides. It is a manifestation of various functions of chitooligosaccharides and one of the important indicators for measuring the quality of chitosan oligosaccharides.
  • the degree of deacetylation of chitooligosaccharides refers to the percentage of the number of sugar residues from which the acetyl group is removed in the total number of sugar residues in chitosan oligosaccharides, and the content of free amino groups is the basis of its various functions.
  • the method of acetyl content is widely used and has become the standard method for the Chinese aquatic industry issued by the Ministry of Agriculture.
  • the determination method using methyl orange or methyl orange-aniline blue as an indicator is used to determine the degree of deacetylation of chitosan oligosaccharide, which has the disadvantage that the color change of the endpoint is not obvious, the reproducibility is poor, and the error is large.
  • the Chinese Pharmacopoeia uses the methyl orange acid base indicator method to determine the chitosan oligosaccharide deacetylation degree error of more than 60%.
  • Nuclear magnetic resonance spectroscopy ( 1 H-NMR) has been included in the US Pharmacopoeia as the gold standard for determining the degree of deacetylation of chitosan.
  • the papers published by Kim et al., "Oligosaccharides and Their Derivatives" and Wang Shixin are different.
  • the mass analysis of the chitosan oligosaccharide product of the source discloses that the degree of deacetylation of chitosan oligosaccharide can be determined by nuclear magnetic resonance spectroscopy, and the error of the measurement result is small.
  • the 1 H-NMR method cannot be widely used because of its high instrument cost and the need for professional technicians.
  • the present invention provides a method for determining the degree of deacetylation of chitooligosaccharides using an acid-base indicator method.
  • the method provided by the invention is a method for accurately and rapidly determining the degree of deacetylation of chitosan oligosaccharides.
  • the method provided by the invention is simple to use, simple and easy to operate, no special pretreatment is required for the sample, and the titration end point is judged obviously, and the measurement is obvious.
  • the error is small and is suitable for quality control in the preparation of chitosan oligosaccharides.
  • the invention provides a method for determining the degree of deacetylation of chitooligosaccharides by using an acid-base indicator method, comprising the following steps:
  • N HCl is the concentration of hydrochloric acid titration solution, mol/L
  • V HCl is the volume of hydrochloric acid titration solution, mL
  • N NaOH is the concentration of sodium hydroxide titration solution, mol/L
  • V NaOH is the volume of sodium hydroxide titration solution, mL
  • G is the weight of the test sample, g
  • W is the weight loss under the weight loss of drying, %
  • 0.016 is the equivalent amount of amino acid of 1 mol / L hydrochloric acid, g
  • 9.94 % is the theoretical amino content.
  • the chitosan oligosaccharide sample has a concentration of 6-10 mg/mL.
  • the concentration of the diluted hydrochloric acid titration solution is 0.1-0.5 mol/L.
  • the indicator is used in an amount of 1% of bromocresol green indicator 1-2 drops.
  • the sodium hydroxide titration solution has a concentration of 0.1 to 0.5 mol/L.
  • the method for determining the degree of deacetylation of chitosan oligosaccharides by the acid-base indicator method provided by the invention can accurately and quickly determine the degree of deacetylation of chitosan oligosaccharides, and the method provided by the invention is simple to use and easy to operate.
  • the sample does not need special pre-treatment, the titration end point is judged obviously, the measurement error is small, suitable Used for quality control in the preparation of chitosan oligosaccharides.
  • the degree of deacetylation of chitosan oligosaccharide was determined by the acid-base indicator method, and the accuracy was high, while the pH of the aqueous solution of the chitosan oligosaccharide was less than 8.0. Error, a pH greater than 8.0 causes a positive error.
  • Figure 1 is a nuclear magnetic resonance spectrum (500 MHz) of chitosan oligosaccharide (COS MW1000 ) having an average molecular weight of ⁇ 1000 Daltons, wherein AE represents a characteristic hydrogen signal at the C2-C6 position on the sugar ring.
  • COS MW1000 chitosan oligosaccharide
  • Figure 2 is a nuclear magnetic resonance spectrum (500 MHz) of chitosan oligosaccharide (COS MW3000 ) having an average molecular weight of ⁇ 3000 Daltons, wherein AE represents a characteristic hydrogen signal at the C2-C6 position on the sugar ring.
  • the raw materials used in the examples of the present invention are all commercially available products, and some of the types and sources of equipment involved are as follows:
  • N HCl is the concentration of hydrochloric acid titration solution, mol/L
  • V HCl is the volume of hydrochloric acid titration solution, mL
  • N NaOH is the concentration of sodium hydroxide titration solution, mol/L
  • V NaOH is the volume of sodium hydroxide titration solution, mL
  • G is the weight of the test sample, g
  • W is the weight loss under the weight loss of drying, %
  • 0.016 is the equivalent amount of amino acid of 1 mol / L hydrochloric acid, g
  • 9.94 % is the theoretical amino content.
  • a 2 represents an integral value of three hydrogen signals of an acetyl group at the C2 acetylamino group on the sugar ring; and A 1 represents an integral value of a hydrogen signal at the C2-C6 position on the sugar ring.
  • the relative error of the determination result of the chitosan oligosaccharide deacetylation degree by the acid-base indicator method of the present invention is less than 0.2%, which is consistent with the measurement result of the 1 H-NMR method.
  • the acid-base indicator method provided by the present invention is used for determining the degree of deacetylation degree of chitosan oligosaccharide.
  • the degree of deacetylation of COS MW1000 and COS MW3000 was determined by referring to the method for determining the degree of deacetylation of chitosan in the Chinese Pharmacopoeia of 2015. The results are shown in Tables 5 and 6.
  • the deacetylation degree of COS MW1000 and COS MW3000 is 30.39 ⁇ 0.70 and 30.32 ⁇ 0.56, respectively, measured by the determination method of chitosan deacetylation degree in the 2015 edition of Chinese Pharmacopoeia.
  • the degree of deacetylation was 93.52 ⁇ 0.13 and 92.81 ⁇ 0.07, and the measurement error was 60% or more.
  • the aqueous solution of chitosan sample is a colorless solution with an isoelectric point of about 3.88.
  • the pH of methyl orange is in the range of 3.1-4.4, and the acid color is red, and the basic color is yellow. Therefore, methyl orange is used as the acid.
  • the alkali indicator is used to determine the degree of deacetylation of chitosan by acid-base titration. The titration end point is obvious, the measurement error is small, and the accuracy is high.
  • chitosan oligosaccharides on the one hand, the aqueous solution of chitosan oligosaccharide sample itself is pale yellow, and methyl orange is used as an indicator, so that the titration end point cannot be accurately judged; on the other hand, the isoelectric value of chitosan oligosaccharide is about 4.80.
  • Exceeding the pH range of methyl orange therefore, the degree of deacetylation of chitosan oligosaccharide measured by the methyl orange acid base indicator method according to the 2015 edition of the Chinese Pharmacopoeia is very large.
  • the pH of bromocresol green is in the range of 3.8-5.4, the acid color is yellow, the basic color is green (blue-green), and bromocresol green is used as the indicator of the acid-base indicator method.
  • the color change of the titration end point is obvious. Accurately determine the endpoint of the titration, which can significantly reduce the measurement error.
  • the inventors of the present invention have found through extensive studies that the pH value of the chitosan oligosaccharide aqueous solution has a great influence on the determination of the degree of deacetylation of chitosan oligosaccharide samples.
  • the inventors of the present invention refer to the method for determining the degree of deacetylation of chitosan in the Chinese Pharmacopoeia of 2015, and determine the degree of deacetylation of COS MW1000 and COS MW3000 by using bromocresol green as an indicator of the acid-base indicator method, and titrating the end point color.
  • the change was remarkable, and the measurement error was remarkably lowered.
  • the measurement error was still 10% or more.
  • the pH value of the chitosan oligosaccharide aqueous solution has a great influence on the determination of the degree of deacetylation of chitosan oligosaccharide samples, which may be due to the combination of acetamido and hydrochloric acid to form chitooligosaccharide hydrochloride in an acidic environment.
  • the acetylamino group cannot be completely released, resulting in a large measurement error.

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Abstract

一种利用酸碱指示剂法测定壳寡糖脱乙酰度的方法包括如下步骤：S1、测定壳寡糖样品中的水分含量作为壳寡糖样品干燥失重项下减失重量；S2、取壳寡糖样品，用蒸馏水溶解后，调节pH值至8.0，加入稀盐酸滴定液，同时滴加1％溴甲酚绿指示剂1-2滴，混匀后用氢氧化钠滴定液滴定至溶液变为绿色即为滴定终点；S3、计算壳寡糖样品的脱乙酰度。该方法是一种准确、快速测定壳寡糖脱乙酰度的方法，同时使用仪器简单，操作简单易行，样品无需特殊的前处理，滴定终点判断明显，测量误差小，适用于壳寡糖制备过程中的质量控制。

Description

一种利用酸碱指示剂法测定壳寡糖脱乙酰度的方法 技术领域
本发明属于化学领域，尤其涉及一种利用酸碱指示剂法测定壳寡糖脱乙酰度的方法。
背景技术
壳寡糖是氨基葡萄糖和N-乙酰氨基葡萄糖以β-1,4糖苷键连接而成的同聚物或异聚物。壳寡糖是壳聚糖的降解产物，但与壳聚糖相比，壳寡糖具有溶解性更好，粘度更低更易被人体吸收的优点，在医药、农业、精细化工等领域有着广泛的应用。壳寡糖不仅具有抗肿瘤、抑菌抗菌、抗氧化等功能，还具有减肥、调脂、增强免疫力等生物功能。
壳寡糖的脱乙酰度能够影响壳寡糖的生物、物理、化学功能与活性，是壳寡糖各种功能的体现，是衡量壳寡糖质量的重要指标之一。壳寡糖的脱乙酰度是指在壳寡糖中总的糖残基数中脱除乙酰基的糖残基数所占的百分比，而游离氨基的含量则是它各种功能的基础。
目前，国内外报道的测定壳聚糖脱乙酰度的方法有很多：包括碱量法(酸碱滴定法、电位滴定法、双突跃滴定法)、红外光谱法、胶体滴定法、折光指数法、破坏样品法(元素分析法、酸水解液相色谱分析与热分析)。甲基橙酸碱指示剂法测定壳聚糖脱乙酰度已收录于2015版《中国药典》，且以甲基橙-苯胺蓝(1∶2)为指示剂的碱量法测定壳聚糖脱乙酰度含量的方法被广泛应用，已成为农业部颁布的中国水产行业标准方法。但采用甲基橙或甲基橙-苯胺蓝为指示剂的测定方法用于测定壳寡糖脱乙酰度时存在着终点颜色变化不明显，重现性差，误差较大的缺点，按照2015版《中国药典》采用甲基橙酸碱指示剂法测定的壳寡糖脱乙酰度误差达到60％以上。核磁共振氢谱(1H-NMR)作为测定壳聚糖脱乙酰度的金标准，已载入了美国药典，同时，Kim等发表的论文“Oligosaccharides and Their Derivatives”和王世欣等发表的论文“不同来源的壳寡糖产品的质量分析”公开了可利用核磁共振氢谱法测定壳寡糖的脱乙酰度，测定结果误差小。但1H-NMR方法由于其仪器成本高且操作需要专业的技术人员，因而无法广泛推广使用。
经检索，现有技术中未见有利用以溴甲酚绿作为指示剂的酸碱指示剂法测定壳寡糖脱乙酰度的报道，也尚未建立壳寡糖脱乙酰度的标准方法。
发明内容
为了解决现有技术中存在的问题，本发明提供了一种利用酸碱指示剂法测定壳寡糖脱乙酰度的方法。本发明提供的方法是一种准确、快速测定壳寡糖脱乙酰度的方法，同时，本发明提供的方法使用仪器简单，操作简单易行，样品无需特殊的前处理，滴定终点判断明显，测量误差小，适用于壳寡糖制备过程中的质量控制。
本发明提供了一种利用酸碱指示剂法测定壳寡糖脱乙酰度的方法，包括如下步骤：
S1、测定壳寡糖样品中的水分含量作为壳寡糖样品干燥失重项下减失重量；
S2、取壳寡糖样品，用蒸馏水溶解后，调节pH值至8.0，加入稀盐酸滴定液，同时滴加0.1-1％的溴甲酚绿指示剂1-6滴，混匀后用氢氧化钠滴定液滴定至溶液变为绿色即为滴定终点；
S3、计算壳寡糖样品的脱乙酰度，公式如下：
Figure PCTCN2017095113-appb-000001
式中，D.D.％为脱乙酰度，％；NHCl为盐酸滴定液的浓度，mol/L；VHCl为盐酸滴定液的体积，mL；NNaOH为氢氧化钠滴定液的浓度，mol/L；VNaOH为氢氧化钠滴定液的体积，mL；G为供试品称重，g；W为干燥失重项下减失重量，％；0.016为1mol/L盐酸相当的氨基量，g；9.94％为理论氨基含量。
进一步地，所述壳寡糖样品的浓度为6-10mg/mL。
进一步地，所述稀盐酸滴定液的浓度为0.1-0.5mol/L。
进一步地，所述指示剂的用量为1％的溴甲酚绿指示剂1-2滴。
进一步地，所述氢氧化钠滴定液浓度为0.1-0.5mol/L。
与现有技术相比，本发明有益效果如下：
(1)本发明提供的利用酸碱指示剂法测定壳寡糖脱乙酰度的方法，可以准确、快速地测定壳寡糖脱乙酰度，且本发明提供的方法使用仪器简单，操作简单易行，样品无需特殊的前处理，滴定终点判断明显，测量误差小，适 用于壳寡糖制备过程中的质量控制。
(2)本发明提供的利用酸碱指示剂法测定壳寡糖脱乙酰度的方法，以溴甲酚绿作为指示剂，滴定终点颜色变化明显，可准确判断滴定终点，降低测定误差。
(3)本发明发明人在研究酸碱指示剂法测定壳寡糖脱乙酰度过程中，通过大量的研究发现，壳寡糖水溶液pH值对样品脱乙酰度的测定具有很大的影响，壳寡糖水溶液中，乙酰氨基在pH 8.0时刚好达到完全游离出来的状态，此时利用酸碱指示剂法测定壳寡糖脱乙酰度，准确度高，而壳寡糖水溶液pH小于8.0时造成负误差，pH大于8.0时造成正误差。
附图说明
图1平均分子量≤1000道尔顿的壳寡糖(COSMW1000)的核磁共振氢谱图(500MHz)，其中，A-E代表糖环上C2-C6位特征氢信号。
图2平均分子量≤3000道尔顿的壳寡糖(COSMW3000)的核磁共振氢谱图(500MHz)，其中，A-E代表糖环上C2-C6位特征氢信号。
具体实施方式
下面通过具体实施例对本发明做进一步的详细说明，且本发明的保护范围不仅仅局限于以下实施例。
本发明实施例中所用原料均为市售产品，其中，涉及的部分设备型号和来源如下：
名称 生产企业
S-25电位pH计 上海雷磁精密科学仪器有限公司
DK-8D型千分之一电子分析天平 德国赛多利斯集团
Sartourius MA150红外水分测定仪 德国赛多利斯集团
Bruker 500MHz核磁共振仪 德国布鲁克公司
实施例1酸碱指示剂法测定壳寡糖脱乙酰度
S1、称取0.5g壳寡糖样品于红外水分测定仪中测定其水分含量，记录结果作为该批次壳寡糖样品干燥失重项下减失重量；
S2、取壳寡糖样品0.5g，精密称定，精密加入50mL蒸馏水，室温下 搅拌使其完全溶解，调pH值至8.0，精密加入0.3mol/L的稀盐酸滴定液18mL，同时滴加1％的溴甲酚绿指示剂1-2滴，混匀后用0.15mol/L的氢氧化钠滴定液滴定至溶液变为绿色即为滴定终点；
S3、计算壳寡糖样品的脱乙酰度，公式如下：
Figure PCTCN2017095113-appb-000002
式中，D.D.％为脱乙酰度，％；NHCl为盐酸滴定液的浓度，mol/L；VHCl为盐酸滴定液的体积，mL；NNaOH为氢氧化钠滴定液的浓度，mol/L；VNaOH为氢氧化钠滴定液的体积，mL；G为供试品称重，g；W为干燥失重项下减失重量，％；0.016为1mol/L盐酸相当的氨基量，g；9.94％为理论氨基含量。
分别对COSMW1000(脱乙酰度≥90％，平均分子量≤1000的壳寡糖样品)、COSMW3000(脱乙酰度≥90％，平均分子量≤3000的壳寡糖样品)进行脱乙酰度测定，结果见表1，表2。
表1 COSMW1000脱乙酰度测定结果
Figure PCTCN2017095113-appb-000003
根据表1可以得知，COSMW1000通过本发明提供的方法，测得其脱乙酰度D.D.值为93.38％，RSD为0.28(n＝6)。
表2 COSMW3000脱乙酰度测定结果
Figure PCTCN2017095113-appb-000004
根据表2可以得知，COSMW3000的壳寡糖样品通过本发明提供的方法，测得其脱乙酰度D.D.值为92.64％，RSD为0.53(n＝6)。
对比例1 1H-NMR法测定脱乙酰度
1)1H-NMR法测定脱乙酰度
分别精密称取20mg COSMW1000和COSMW3000溶解在5mL的D2O(99.96％)中，得浓度为4mg/mL的样品溶液，将所得样品溶液转移至8mm核磁管中进行测定，共振频率为500MHz，测定温度为297K，最后对目标信号积分，其中糖环上C2位乙酰胺基中乙酰基的氢信号(Acetyl-H)在1.9-2.1ppm，糖环上C2-C6位氢信号在2.6-6.0ppm处。COSMW1000，COSMW3000核磁共振氢谱图分别见图1，图2。根据表3，对核磁共振氢谱图对应峰进行积分，脱乙酰度计算公式为：
D.D(％)＝{1-[(7*A2)/(3*A1)]}*100
其中，A2代表糖环上C2位乙酰氨基中乙酰基的3个氢信号的积分值；A1代表糖环上C2-C6位氢信号的积分值。
表3 25℃下壳寡糖氘水溶液氢质子化学位移
Figure PCTCN2017095113-appb-000005
利用1H-NMR法测得COSMW1000，COSMW3000对应的脱乙酰度测定结果分别为93.52±0.13(n＝6)，92.81±0.07(n＝6)。
2)1H-NMR法与本发明酸碱指示剂法测定结果的对比
1H-NMR法与本发明酸碱指示剂法测定结果的对比结果见表4。
表4 1H-NMR与本发明酸碱指示剂法测定结果对比(n＝6)
样品 1H-NMR(％) 溴甲酚绿指示剂法(％) 相对误差
COSMW1000 93.52±0.13 93.38±0.28 0.14±0.15
COSMW3000 92.81±0.07 92.64±0.53 0.17±0.46
从表4结果可以看出，本发明酸碱指示剂法测定壳寡糖脱乙酰度的测定结果相对误差小于0.2％，与1H-NMR法的测定结果一致。说明本发明提供的酸碱指示剂法用于测定壳寡糖脱乙酰度准确度高。
对比例2甲基橙酸碱指示剂法测定壳寡糖脱乙酰度
参照2015版《中国药典》中壳聚糖脱乙酰度的测定方法测定COSMW1000和COSMW3000的脱乙酰度，测定结果如表5和表6所示。
表5 COSMW1000脱乙酰度测定结果
Figure PCTCN2017095113-appb-000006
表6 COSMW3000脱乙酰度测定结果
Figure PCTCN2017095113-appb-000007
由表5和表6的结果可知，利用2015版《中国药典》中壳聚糖脱乙酰度的测定方法测得COSMW1000、COSMW3000对应的脱乙酰度分别为 30.39±0.70、30.32±0.56，与利用1H-NMR法测得的COSMW1000、COSMW3000对应的脱乙酰度93.52±0.13、92.81±0.07相比，具有60％以上的测定误差。
壳聚糖样品水溶液是无色溶液，其等电点在3.88左右，甲基橙pH变色范围在3.1-4.4，且酸式色为红色，碱式色为黄色，因此，以甲基橙作为酸碱指示剂利用酸碱滴定法测定壳聚糖的脱乙酰度，滴定终点明显，测量误差小，准确度高。而对于壳寡糖而言，一方面，壳寡糖样品水溶液本身呈现淡黄色，采用甲基橙作为指示剂，使得滴定终点无法准确判断；另一方面，壳寡糖等电点值在4.80左右，超出甲基橙pH变色范围，因此，按照2015版《中国药典》采用甲基橙酸碱指示剂法测定的壳寡糖脱乙酰度误差很大。溴甲酚绿pH变色范围在3.8-5.4，酸式色为黄色，碱式色为绿色(蓝绿色)，采用溴甲酚绿作为酸碱指示剂法的指示剂，滴定终点颜色变化明显，可准确判断滴定终点，从而可显著降低测定误差。
对比例3酚酞酸碱指示剂法测定壳寡糖脱乙酰度
参考2015版《中国药典》中壳聚糖脱乙酰度的测定方法，以酚酞作为指示剂测定COSMW1000和COSMW3000的脱乙酰度，测定结果如表7和表8所示。
表7 COSMW1000脱乙酰度测定结果
Figure PCTCN2017095113-appb-000008
表8 COSMW3000脱乙酰度测定结果
Figure PCTCN2017095113-appb-000009
由表7和表8的结果可知，以酚酞作为指示剂测得COSMW1000、COSMW3000对应的脱乙酰度分别为66.72±0.23、62.01±0.29，与利用1H-NMR法测得的COSMW1000、COSMW3000对应的脱乙酰度93.52±0.13、92.81±0.07相比，存在30％左右的测定误差。
对比例4 pH的考察
本发明发明人在研究过程中，通过大量的研究发现，壳寡糖水溶液pH值对壳寡糖样品脱乙酰度的测定具有很大的影响。
本发明发明人参考2015版《中国药典》中壳聚糖脱乙酰度的测定方法，采用溴甲酚绿作为酸碱指示剂法的指示剂测定COSMW1000和COSMW3000的脱乙酰度，滴定终点颜色变化明显，从而显著降低了测定误差，但与1H-NMR法的测定方法相比，仍具有10％以上的测定误差。
研究过程中发现，受壳寡糖制备工艺的影响，市售壳寡糖的水溶液的pH值为5.12左右，呈现弱酸性。壳寡糖是一种碱性氨基多糖，若其水溶液呈现弱酸性，利用酸碱指示剂法测定其脱乙酰度时，必定会引起测定误差。
在pH值改变，其他测定条件和方法与实施例1相同的条件下考察pH对酸碱指示剂法测定壳寡糖脱乙酰度的测定结果的影响，结果见表9。
表9壳寡糖水溶液不同pH值对壳寡糖脱乙酰度测定结果的影响(n＝6)
pH 7.60 7.80 7.90 8.00 8.10 8.20
D.DMW1000(％) 87.45±0.18 90.94±0.21 91.64±0.31 93.44±0.13 95.62±0.20 97.65±0.25
D.DMW3000(％) 88.14±0.19 89.86±0.20 90.82±0.16 92.69±0.11 94.25±0.18 97.12±0.23
根据表9的结果可知，壳寡糖水溶液pH值对壳寡糖样品脱乙酰度的测定具有很大的影响，原因可能是在酸性环境中乙酰氨基与盐酸结合形成了壳寡糖盐酸盐，使乙酰氨基不能完全游离出来，从而造成了较大的测定误差。随着溶液酸碱性的变化，乙酰氨基慢慢游离出来，在pH＝8.00时达到刚好完全游离出来的状态，pH<8.00造成负误差，pH>8.00造成正误差。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (5)

  1. 一种利用酸碱指示剂法测定壳寡糖脱乙酰度的方法，其特征在于：包括如下步骤：
    S1、测定壳寡糖样品中的水分含量作为壳寡糖样品干燥失重项下减失重量；
    S2、取壳寡糖样品，用蒸馏水溶解后，调节pH值至8.0，加入稀盐酸滴定液，同时滴加0.1-1％的溴甲酚绿指示剂1-6滴，混匀后用氢氧化钠滴定液滴定至溶液变为绿色即为滴定终点；
    S3、计算壳寡糖样品的脱乙酰度，公式如下：
    Figure PCTCN2017095113-appb-100001
    式中，D.D.％为脱乙酰度，％；NHCl为盐酸滴定液的浓度，mol/L；VHCl为盐酸滴定液的体积，mL；NNaOH为氢氧化钠滴定液的浓度，mol/L；VNaOH为氢氧化钠滴定液的体积，mL；G为供试品称重，g；W为干燥失重项下减失重量，％；0.016为1mol/L盐酸相当的氨基量，g；9.94％为理论氨基含量。
  2. 根据权利要求1所述的利用酸碱指示剂法测定壳寡糖脱乙酰度的方法，其特征在于：所述壳寡糖样品的浓度为6-10mg/mL。
  3. 根据权利要求1所述的利用酸碱指示剂法测定壳寡糖脱乙酰度的方法，其特征在于：所述稀盐酸滴定液的浓度为0.1-0.5mol/L。
  4. 根据权利要求1所述的利用酸碱指示剂法测定壳寡糖脱乙酰度的方法，其特征在于：所述指示剂的用量为1％的溴甲酚绿指示剂1-2滴。
  5. 根据权利要求1所述的利用酸碱指示剂法测定壳寡糖脱乙酰度的方法，其特征在于：所述氢氧化钠滴定液浓度为0.1-0.5mol/L。
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