CN114113352A - 发酵液中内酯型槐糖酯含量的测定方法 - Google Patents
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Abstract
本发明所提出的发酵液中内酯型槐糖脂含量的测定方法；因为化妆品中的槐糖脂含量是决定卸妆难易程度的重要因素，所以在化妆品生产和检验环节都需要快速的检测出槐糖脂的含量；故而本发明首次建立了测定发酵液中内酯型槐糖脂含量的测定方法；采用超高效液相色谱分析，结合蒸发光散射检测器进行检测能够对槐糖脂进行定量测定；最优的色谱条件为AQ‑C18色谱柱，色谱柱温度为25℃，进样量10μL；流动相包括有A相和B相，A相为乙腈，B相为体积百分比为5:95的甲醇：0.01M乙酸铵溶液；流速为0.65mL/min；并采用梯度洗脱的方式进行洗脱；ELSD检测器的条件为：漂移管温度90℃，增益300，气体流量32psi，喷雾器处于冷却状态，该测定方法能够准确、快速地测定出槐糖脂的含量。
Description
技术领域
本发明涉及槐糖酯的定量分析技术领域，尤其涉及一种发酵液中内酯型槐糖酯含量的测定方法。
背景技术
槐糖脂是一种糖脂类生物表面活性剂。其具有常规表面活性剂所具有的增溶、乳化、润湿、发泡、分散、降低表面张力等通用性能，而且还具有无毒、100％可生物降解、耐温、耐高盐、适应pH范围广及对环境友好等特性。槐糖脂可应用于石油、环保、医药、食品、化妆品、洗涤、家居护理、农业、饲料等领域，可达到部分或完全替代化学合成表面活性剂使用。槐糖脂作为生物表面活性剂的一种，具有较好的表面性能、低毒、生物可降解以及对环境无污染等诸多优点，且在化妆品的应用中，槐糖脂的含量大小也是决定卸妆难易程度的重要因素，所以在槐糖脂的研发、生产和验收环节中都需要快速的检测出槐糖脂的含量；故而本发明首次建立了测定发酵液中内酯型槐糖脂含量的测定方法，并且化妆品卸妆效果的好坏也是取决于内酯型槐糖酯的含量高低。因此，在一定意义上来说内酯型槐糖酯的含量测定是一个验证化妆品质量的重要参数之一；但是在现有技术中，通常都是采用HPLC/TOF/MS法对槐糖脂进行定性的测定，未有提到如何测定内酯型槐糖酯；所以如何快速准确的测定内酯型槐糖酯含量是一个亟需解决的问题。
发明内容
针对上述技术中存在的如:还未有一种高效、快速测定发酵液中内酯型槐糖酯含量的测定方法。
具体为一种发酵液中内酯型槐糖酯含量的测定方法，包括以下步骤:
(1)标准工作溶液配制
称取槐糖酯标准品，并采用50％乙醇水溶液作为溶剂定容，得到浓度为15mg/mL的溶液A；再采用50％乙醇水溶液稀释溶液A得到0.75mg/mL、1.5mg/mL、7.5mg/mL、12mg/mL的梯度浓度标准工作液；
(2)待测品制备
称取待测品，用50％乙醇水溶液稀释得到浓度为100mg/mL的溶液C，然后进行超声萃取后进行离心处理，最后用离心后的溶液经微孔滤膜过滤得到滤液；
(3)液相色谱分析
采用超高效合相色谱仪对步骤(1)得到的梯度浓度标准工作液和步骤(2)得到的滤液进行分析，利用ELSD检测器进行检测；
色谱条件：采用AQ-C18柱(4.6mm×250mm，5μm)；色谱柱温度为25℃，进样量10μL；流动相包括有A相和B相，所述A相为乙腈，B相为体积百分比为5:95的甲醇：0.01M乙酸铵溶液；流速为0.65mL/min；梯度洗脱条件为：
0min-10min：A相与B相的体积百分比由5％：95％逐步递增至16％：84％，
10min-15min：A相与B相的体积百分比达到16％：84％时保留5min持续洗脱5min；
15min-16min：A相与B相的体积百分比由16％：84％逐步递减至5％：95％；
ELSD检测器的条件为：漂移管温度90℃，增益300，气体流量32psi，喷雾器处于冷却状态。
作为优选，在步骤(2)中，采用40KHz频率的超声波，在26℃温度下超声10分钟。
作为优选，在步骤(2)中，称取超声后的溶液10mL置于离心管中，在8000r/min下离心10min，取5mL经微孔滤膜过滤。
作为优选，所述微孔滤膜为0.20μm。
作为优选，在步骤(3)中，所述0.01M乙酸铵溶液利用甲酸调配pH为4.75。
作为优选，所述槐糖酯标准品的纯度≥97％。
本发明的有益效果是:本发明所提出的发酵液中内酯型槐糖脂含量的测定方法；因为化妆品中的槐糖脂含量是卸妆难易程度的重要影响因素，所以在化妆品生产和检验环节都需要快速的检测出槐糖脂的含量；故而本发明首次建立了测定发酵液中内酯型槐糖脂含量的测定方法；采用超高效液相色谱分析，结合蒸发光散射检测器进行检测能够对槐糖脂进行定量测定；最优的色谱条件为AQ-C18色谱柱，色谱柱温度为25℃，进样量10μL；流动相包括有A相和B相，所述A相为乙腈，B相为体积百分比为5:95的甲醇：0.01M乙酸铵溶液；流速为0.65mL/min；梯度洗脱条件为：0min-10min：A相与B相的体积百分比由5％：95％逐步递增至16％：84％，10min-15min：A相与B相的体积百分比达到16％：84％时保留5min持续洗脱5min；15min-16min：A相与B相的体积百分比由16％：84％逐步递减至5％：95％；ELSD检测器的条件为：漂移管温度90℃，增益300，气体流量32psi，喷雾器处于冷却状态，该测定方法能够准确、快速地测定出槐糖脂的含量。
附图说明
图1为本发明内酯型槐糖脂标准品的液相色谱图；
图2为本发明1号发酵液样品的液相色谱图；
图3为本发明2号发酵液样品的液相色谱图；
图4为本发明3号发酵液样品的液相色谱图；
图5为本发明4号发酵液样品的液相色谱图；
图6为本发明5号发酵液样品的液相色谱图；
图7为本发明6号发酵液样品的液相色谱图。
具体实施方式
为了更清楚地表述本发明，下面结合实施例对本发明作进一步地描述。
首先，对于本发明的发明初衷和思路，是因为槐糖脂的应用十分广泛，比如在化妆品中就需要用到内酯型槐糖脂，并且内酯型槐糖脂的含量是决定化妆品卸妆效果好坏的重要指标之一，所以对于化妆品的制备环节和出厂检验环节都需要对内酯型槐糖脂做一个定量的测定，但是现在的测试方法中，都未提及如何对内酯型槐糖脂的含量进行准确测定，而现有技术中提到的HPLC/TOF/MS均是对定性分析有优势，定量测定不精准的方法；所以需要一种测定方法能够又快又准确的测定出待测物中内酯型槐糖脂的含量。
蒸发光散射检测器是一种通用型的检测器,可检测挥发性低于流动相的任何样品,而不需要样品含有发色基团。蒸发光散射检测器灵敏度比示差折光检测器高,对温度变化不敏感,基线稳定,适合与梯度洗脱液相色谱联用。蒸发光散射检测器已被广泛应用于碳水化合物、类脂、脂肪酸和氨基酸、药物以及聚合物等的检测。ELSD的响应值与样品质量成正比，因此可以用来确定样品的纯度或探测未知物体。蒸发光散射(ELS)检测的原理是，雾化来自超高效液相色谱系统的溶剂，并将产生的液滴夹杂在气流中。然后流动相从液滴中蒸发出来。分析物的挥发性低于流动相时，它作为“干”溶质颗粒留在气流中并流到ELS检测器中。到达检测器后，颗粒使光束发生散射。散射光的量可以测量出来，并且与洗脱物质的浓度有关。
实施例
(1)标准工作溶液配制
称取槐糖酯标准品(槐糖酯标准品的纯度≥97％，精确至0.0001g)，并采用50％乙醇水溶液作为溶剂定容，得到浓度为15mg/mL的溶液A；再采用50％乙醇水溶液稀释溶液A得到0.75mg/mL、1.5mg/mL、7.5mg/mL、12mg/mL的梯度浓度标准工作液；
(2)待测品制备
称取待测品1.000g于10mL容量瓶中，用50％乙醇水溶液稀释得到浓度为100mg/mL的溶液C，然后进行40KHz频率的超声波，在26℃温度下超声萃取10分钟；并称取超声后的溶液10mL置于离心管中，在8000r/min下离心10min，取5mL经微孔滤膜过滤；
(3)液相色谱分析
采用超高效合相色谱仪对步骤(1)得到的梯度浓度标准工作液和步骤(2)得到的滤液进行分析，利用ELSD检测器进行检测；
色谱条件：采用AQ-C18柱(4.6mm×250mm，5μm)；色谱柱温度为25℃，进样量10μL；流动相包括有A相和B相，A相为乙腈，B相为体积百分比为5:95的甲醇：0.01M乙酸铵溶液(利用甲酸调配pH为4.75)，pH调节过程为：称取0.771g乙酸铵至1000ml水(超纯水)中溶解，并用0.1％甲酸调PH值至4.75，调节pH是为了让内酯型槐糖脂与其它杂质能够在有效分离，在色谱图上不会出现重叠的峰，可以清晰的区分和观察到色谱图上待测样中各物质的出峰；流速为0.65mL/min；梯度洗脱条件为：
0min-10min：A相与B相的体积百分比由5％：95％逐步递增至16％：84％，
10min-15min：A相与B相的体积百分比达到16％：84％时保留5min持续洗脱5min；
15min-16min：A相与B相的体积百分比由16％：84％逐步递减至5％：95％；并且在实际检测过程中，且平衡10min后进下一针。
ELSD检测器的条件为：漂移管温度90℃，增益300，气体流量32psi，喷雾器处于冷却状态。
ELSD的参数选择中，气体流量越低形成的液滴越大，液滴越大则散射的光越多，从而提高了分析灵敏度，但是越大的液滴在漂移管中越难蒸发。最后发现在气体流量为32psi时信噪比最优，同时高有机含量流动相比高含水量流动相要求蒸发的漂移管温度低。同时发现漂移管温度为90℃时，可以获得最佳灵敏度。以达到稳定地、具有优异分离性的峰。
将上述步骤(1)中的各浓度梯度的槐糖脂标准溶液按照方法流程进行测定；同时在市场上随机购买了六个批次的发酵液进行内酯型槐糖脂含量的测定；得到如图1-图7的液相色谱图；根据内酯型槐糖脂标准品的液相色谱图的出峰时间可以知道，在4.236分钟左右内酯型槐糖脂可以被分离检测到；所以在图2-图7可以判断出内酯型槐糖脂的反应峰和其它杂质出峰，从而利用峰面积进行含量的测定；六批次发酵液样品中内酯型槐糖脂的含量如下表：
表1
| 名称 | 内酯型槐糖脂含量/％ |
| 1号发酵液样品 | 0.16 |
| 2号发酵液样品 | 10.17 |
| 3号发酵液样品 | 34.76 |
| 4号发酵液样品 | 41.37 |
| 5号发酵液样品 | 76.64 |
| 6号发酵液样品 | 79.85 |
本发明的优势在于:
1)本发明建立了一种利用超高效合相色谱串接蒸发光散射检测器快速检测发酵液中内酯型槐糖脂含量的测定方法，能够准确地进行定量，为发酵液中内酯型槐糖脂的准确判定、快速检测提供科学依据；
2)本发明中的处理流程选择对发酵液中内酯型槐糖脂达到了优异的分离效果；
3)实现在5分钟内，对样品的快速灵敏分析。
以上公开的仅为本发明的几个具体实施例，但是本发明并非局限于此，任何本领域的技术人员能思之的变化都应落入本发明的保护范围。
Claims (6)
1.一种发酵液中内酯型槐糖酯含量的测定方法，其特征在于，包括以下步骤:
(1)标准工作溶液配制
称取槐糖酯标准品，并采用50％乙醇水溶液作为溶剂定容，得到浓度为15mg/mL的溶液A；再采用50％乙醇水溶液稀释溶液A得到0.75mg/mL、1.5mg/mL、7.5mg/mL、12mg/mL的梯度浓度标准工作液；
(2)待测品制备
称取待测品，用50％乙醇水溶液稀释得到浓度为100mg/mL的溶液C，然后进行超声萃取后进行离心处理，最后用离心后的溶液经微孔滤膜过滤得到滤液；
(3)液相色谱分析
采用超高效合相色谱仪对步骤(1)得到的梯度浓度标准工作液和步骤(2)得到的滤液进行分析，利用ELSD检测器进行检测；
色谱条件：采用AQ-C18柱(4.6mm×250mm，5μm)；色谱柱温度为25℃，进样量10μL；流动相包括有A相和B相，所述A相为乙腈，B相为体积百分比为5:95的甲醇：0.01M乙酸铵溶液；流速为0.65mL/min；梯度洗脱条件为：
0min-10min：A相与B相的体积百分比由5％：95％逐步递增至16％：84％，
10min-15min：A相与B相的体积百分比达到16％：84％时保留5min持续洗脱5min；
15min-16min：A相与B相的体积百分比由16％：84％逐步递减至5％：95％；
ELSD检测器的条件为：漂移管温度90℃，增益300，气体流量32psi，喷雾器处于冷却状态。
2.根据权利要求1所述的发酵液中内酯型槐糖酯含量的测定方法，其特征在于，在步骤(2)中，采用40KHz频率的超声波，在26℃温度下超声10分钟。
3.根据权利要求2所述的发酵液中内酯型槐糖酯含量的测定方法，其特征在于，在步骤(2)中，称取超声后的溶液10mL置于离心管中，在8000r/min下离心10min，取5mL经微孔滤膜过滤。
4.根据权利要求3所述的发酵液中内酯型槐糖酯含量的测定方法，其特征在于，所述微孔滤膜为0.20μm。
5.根据权利要求1所述的发酵液中内酯型槐糖酯含量的测定方法，其特征在于，在步骤(3)中，所述0.01M乙酸铵溶液利用甲酸调配pH为4.75。
6.根据权利要求1所述的发酵液中内酯型槐糖酯含量的测定方法，其特征在于，所述槐糖酯标准品的纯度≥97％。
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| GB201009882D0 (en) * | 2010-06-11 | 2010-07-21 | Univ Gent | Yeast strains modified in their sophorolipid production and uses thereof |
| WO2012167813A1 (en) * | 2011-06-06 | 2012-12-13 | Ecover Co-Ordination Center N.V. | Improved sophorolactone production |
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