CN114650882A - 用于混合模式阴离子交换反相液相色谱的材料和方法 - Google Patents
用于混合模式阴离子交换反相液相色谱的材料和方法 Download PDFInfo
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- CN114650882A CN114650882A CN202080077828.0A CN202080077828A CN114650882A CN 114650882 A CN114650882 A CN 114650882A CN 202080077828 A CN202080077828 A CN 202080077828A CN 114650882 A CN114650882 A CN 114650882A
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Abstract
在各个方面,本公开涉及包括色谱表面的高纯度色谱材料,其中该色谱表面包含疏水改性剂和包含在电离时带正电的一个或多个阴离子交换部分的可电离改性剂,并且涉及含有此类材料的装置。在其他方面,本公开提供了用于混合模式阴离子交换反相液相色谱的方法,该方法包括:(a)将包含多种酸性分析物(例如,酸性聚糖)的样品装载到包含这种高纯度色谱材料的色谱分离装置上,以及(b)用包含水、有机溶剂和有机酸盐的流动相从该高纯度色谱材料洗脱吸附的酸性分析物,其中在洗脱过程中,该流动相的pH、该流动相的离子强度和该有机溶剂的浓度随时间推移而改变。
Description
相关申请的交叉引用
本申请要求于2019年11月7日提交的美国临时专利申请号62/932,174的权益和优先权,该临时专利申请的全部内容据此以引用方式并入。
背景技术
聚糖在整个生物系统中以游离状态以及作为糖蛋白、糖脂和蛋白聚糖的一部分以缀合形式存在。聚糖在各种生物和生理过程中发挥作用。
利用MS的分析在聚糖和氨基酸分析以及蛋白质组学方面已达到高度发展。现有技术利用反应快速并且提供良好MS/荧光信号的标记分子。荧光检测是确定不同聚糖的相对量的非常有用的工具。另一方面,MS用于确定分子信息。因此,期望具有两种检测形式。用于现有技术的聚糖检测的选择标签在相对高的疏水性与高pKa可电离残基配对时往往是独特的,使得它们成为所谓的“两亲强碱性”部分。
持续需要产生对聚糖(包括用两亲强碱性部分标记的聚糖)的最佳选择性和分离度的高纯度色谱材料和相应的分离方法。
发明内容
在各个方面,本公开利用了包括色谱表面的高纯度色谱材料,其中该色谱表面包含疏水改性剂和包含在电离时带正电的一个或多个阴离子交换部分的可电离改性剂。另外,高纯度色谱材料在约3至约10的pH范围内是水解稳定的,并且在一些实施方案中,有利地在约2至约12的pH范围内是稳定的。
在一些实施方案中,高纯度色谱材料是无机材料、杂化有机/无机材料、具有杂化表面层的无机材料、具有无机表面层的杂化材料或具有不同杂化表面层的杂化材料。
在高纯度色谱材料包含无机材料的各种实施方案中,无机材料可以选自二氧化硅、氧化铝、二氧化钛、氧化锆以及它们的组合等。
在高纯度色谱材料包含杂化有机/无机材料的各种实施方案中,该材料可以包含≡Si-(CH2)n-Si≡部分和/或≡Si–O–(CH2)mCH3部分,其中n是等于1、2、3或4的整数,并且m是等于0、1、2或3的整数。一种包含≡Si-(CH2)n-Si≡部分和≡Si-O-(CH2)mCH3部分(并且Et表示-CH2CH3)两者的这样的材料在下式I中示意性地示出(其中n表示重复结构):
在各种实施方案中,高纯度色谱材料可以通过水解缩合一种或多种硅烷化合物来形成,所述一种或多种硅烷化合物通常包括(a)式SiZ1Z2Z3Z4的一种或多种硅烷化合物,其中Z1、Z2、Z3和Z4独立地选自Cl、Br、I、C1-C4烷氧基、C1-C4烷基氨基和C1-C4烷基,但是Z1、Z2、Z3和Z4中的至多三个可以是C1-C4烷基,例如四烷氧基硅烷,包括四-C1-C4-烷氧基硅烷诸如四甲氧基硅烷、四乙氧基硅烷、四氯硅烷、甲基-三乙氧基硅烷和甲基-三氯硅烷等,以及烷基-三烷氧基硅烷,例如C1-C4-烷基-三-C1-C4-烷氧基硅烷,诸如甲基三乙氧基硅烷、甲基三甲氧基硅烷或乙基三乙氧基硅烷等,和/或(b)式Si Z1Z2Z3—R—SiZ4Z5Z6的一种或多种化合物,其中Z1、Z2和Z3独立地选自Cl、Br、I、C1-C4烷氧基、C1-C4烷基氨基和C1-C4烷基,但是Z1、Z2和Z3中的至多两个可以是C1-C4烷基,并且其中Z4、Z5和Z6独立地选自Cl、Br、I、C1-C4烷氧基、C1-C4烷基氨基和C1-C4烷基,但是Z4、Z5和Z6中的至多两个可以是C1-C4烷基,并且其中R是有机基团,例如,选自C1-C18亚烷基、C2-C18亚烯基、C2-C18亚炔基或C6-C18亚芳基基团,其示例包括双(三烷氧基甲硅烷基)烷烃,通常为双(三-C1-C4-烷氧基甲硅烷基)C1-C4-烷烃诸如双(三甲氧基甲硅烷基)甲烷、双(三甲氧基甲硅烷基)乙烷、双(三乙氧基甲硅烷基)甲烷、双(三乙氧基甲硅烷基)乙烷等。
因此,在一些实施方案中,高纯度色谱材料可以通过水解缩合包含一种或多种烷氧基硅烷化合物的一种或多种有机硅烷化合物来形成。烷氧基硅烷化合物的示例包括例如四烷氧基硅烷(例如,四甲氧基硅烷(TMOS)、四乙氧基硅烷(TEOS)等)、烷基烷氧基硅烷诸如烷基三烷氧基硅烷(例如,甲基三甲氧基硅烷、甲基三乙氧基硅烷(MTOS)、乙基三乙氧基硅烷等)和双(三烷氧基甲硅烷基)烷烃(例如,双(三甲氧基甲硅烷基)甲烷、双(三甲氧基甲硅烷基)乙烷、双(三乙氧基甲硅烷基)甲烷、双(三乙氧基甲硅烷基)乙烷(BTE)等),以及前述的组合。在这些实施方案中的某个中,基于二氧化硅的材料可由两种烷氧基硅烷化合物制备:四烷氧基硅烷,诸如TMOS或TEOS,和烷基烷氧基硅烷诸如MTOS,或双(三烷氧基甲硅烷基)烷烃,诸如BTEE。当BTEE用作单体时,所得材料是有机-无机杂化材料,其有时被称为乙烯桥连杂化(BEH)材料,并且可提供优于常规基于硅胶的材料的各种优点,包括化学稳定性和机械稳定性。上文的式I是BEH材料的示意图,其可以由TEOS和BTEE的水解缩合形成。
如上所述,在本公开中,高纯度色谱材料诸如上文所描述的那些等具有色谱表面,其中该色谱表面包含疏水改性剂和在电离时带正电的一个或多个阴离子交换部分。
在一些实施方案中,疏水改性剂包含具有4至30个碳原子的烃部分。
在一些实施方案中,疏水改性剂包含C4-C30脂族部分(例如,直链、支链或环状C4-C30烷基部分)、C4-C30芳族部分、苯基烷基(例如,苯基己基等)部分或氟芳族(例如,五氟苯基烷基等)部分。
在一些实施方案中,疏水改性剂包含一个或多个烷基基团,包括含有4至30个碳原子、通常8至18个碳原子的直链、支链和环状烷基基团。
在各种实施方案中,色谱表面通过使高纯度色谱材料与反应性疏水改性试剂反应而衍生,该应性疏水改性试剂例如包含(a)疏水部分和(b)一个或多个反应性硅烷基团的反应性疏水改性试剂。
在某些实施方案中,可电离改性试剂具有式M(SiZ1Z2Z3)n,其中n=1、2、3或更大,M表示疏水部分,并且Z1、Z2和Z3独立地选自Cl、Br、I、C1-C4烷氧基和C1-C4烷基氨基。疏水部分的示例包括具有4至30个碳原子的烃部分、包含C4-C30脂族部分、C4-C30芳族部分、苯基烷基部分或氟芳族部分的烃部分。在一些实施方案中,疏水改性剂包含一个或多个烷基基团,包括含有4至30个碳原子、通常8至18个碳原子的直链、支链和环状烷基基团。
在某些实施方案中,可电离改性试剂可以是反应性有机硅烷,诸如选自苯基己基三氯硅烷、五氟苯基丙基三氯硅烷、辛基三氯硅烷、十八烷基三氯硅烷、辛基二甲基氯硅烷和十八烷基二甲基氯硅烷等的反应性C4-C30有机硅烷。
在一些实施方案中,疏水改性剂以0.1微摩尔/平方米至5微摩尔/平方米范围内的表面浓度存在。
关于可电离改性剂,在一些实施方案中,可电离改性剂包含pKa范围为4至13的阴离子交换部分。
在一些实施方案中,可电离改性剂包含阴离子交换部分,该阴离子交换部分选自含胺部分、含胍部分、含脒部分、含吡啶基部分、含咪唑基部分、含咔唑基部分、含异氰脲酸酯部分和/或含氨基脲基部分,其通过一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个或更多个甲硅烷氧基键连接到色谱表面,并且在某些有益的实施方案中,通过六个或更多个甲硅烷氧基键连接到色谱表面。例如,可电离改性剂可以是通过六个甲硅烷氧基键连接到色谱表面的含胺部分,其可以由双(三烷氧基甲硅烷基)胺可电离改性试剂形成,或者可以是通过九个甲硅烷氧基键连接到色谱表面的含胺部分,其可以由三(三烷氧基甲硅烷基)胺可电离改性试剂形成,以及其他许多选项。
在各种实施方案中,可电离改性剂包含阴离子交换部分,该阴离子交换部分选自含胺部分、含胍部分、含脒部分、含吡啶基部分、含咪唑基部分、含咔唑基部分、含异氰脲酸酯部分和/或含氨基脲基部分,该阴离子交换部分桥接两个或更多个甲硅烷氧基基团,每个甲硅烷氧基基团通过三个甲硅烷氧基键连接到色谱表面。
在本公开的一些方面,疏水改性剂:可电离改性剂的摩尔比为约2:1至约100:1;约2.5:1至约67:1;约4:1至约35:1;约5:1至约25:1。
在其他方面,可电离改性剂的表面浓度为约0.01μmol/m2至约1.0μmol/m2、约0.03μmol/m2至约0.5μmol/m2或约0.1μmol/m2至约0.3μmol/m2。
在各种实施方案中,色谱表面可以通过使高纯度色谱材料与可电离改性试剂反应而衍生。在各种实施方案中,可电离改性试剂可以包含(a)选自含胺部分、含胍部分、含脒部分、含吡啶基部分、含咪唑基部分、含咔唑基部分、含异氰脲酸酯部分和/或含氨基脲基部分的一个或多个部分和(b)一个或多个反应性硅烷基团。
含胺部分可以包含例如一个、两个、三个、四个或更多个氨基基团,例如选自伯胺基团、仲胺基团、叔胺基团以及它们的组合。类似地,含胍部分、含脒部分、含吡啶基部分、含咪唑基部分、含咔唑基部分、含异氰脲酸酯部分或含氨基脲基部分可以分别含有一个、两个、三个或更多个胍基团、脒基团、吡啶基基团、咪唑基基团、咔唑基基团、异氰尿酸酯基团或氨基脲基基团。
反应性硅烷基团可以含有一个、两个或三个反应性基团,通常含有三个反应性基团,其可以选自例如Cl、Br、I、C1-C4烷氧基和C1-C4烷基氨基。
在某些实施方案中,可电离改性试剂具有式A(SiZ1Z2Z3)n,其中n=1、2、3、4或更大,A表示含胺部分、含胍部分、含脒部分、含吡啶基部分、含咪唑基部分、含咔唑基部分、含异氰脲酸酯部分和/或含氨基脲基部分,并且Z1、Z2和Z3独立地选自Cl、Br、I、C1-C4烷氧基、C1-C4烷基氨基和C1-C4烷基,但是Z1、Z2和Z3中的至多两个可以是C1-C4烷基,并且在各种实施方案中,Z1、Z2和Z3都不是C1-C4烷基(换句话说,Z1、Z2和Z3独立地选自Cl、Br、I、C1-C4烷氧基和C1-C4烷基氨基)。当n=2或更大时,-SiZ1Z2Z3基团中的每一个可以彼此相同,或者-SiZ1Z2Z3基团中的每一个可以彼此不同。
在某些实施方案中,可电离改性试剂选自双甲硅烷基可电离改性剂和三甲硅烷基可电离改性剂。
具有式A(SiZ1Z2Z3)n的具体可电离改性试剂可以选自以下等等中的一种或多种:N-(2-氨基乙基)-11-氨基十一烷基三甲氧基硅烷(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);1-(三乙氧基甲硅烷基)-N,N-双[(三乙氧基甲硅烷基)甲基]-甲胺(n=3,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);2,2-二甲基-4-(三甲氧基甲硅烷基)1-丁胺(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N-甲基-3-(三甲氧基甲硅烷基)-N-[3-(三甲氧基甲硅烷基)丙基]-1-丙胺(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N,N-二甲基-6-(三甲氧基甲硅烷基)-1-己胺(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);2-[双[3-(三甲氧基甲硅烷基)丙基]氨基]-乙醇(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);7-(2-羟乙基)-3,3-二甲氧基-10-[3-(三甲氧基甲硅烷基)丙基]2-氧杂-7,10-二氮杂-3-硅杂十二烷-12-醇(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);4-(二乙氧基乙基甲硅烷基)-N-[4-(二乙氧基乙基甲硅烷基)丁基]-1-丁胺(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);11-氨基十一烷基三乙氧基硅烷(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);4-氨基丁基三乙氧基硅烷(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N1,NF2-双[(二乙氧基甲基甲硅烷基)甲基]-N1,N2-二甲基-1,2-乙二胺(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N1-(3-苯基-2-丙烯-1-基)-N1-[3-(三甲氧基甲硅烷基)丙基]-1,2-乙二胺(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N-乙基-2-甲基-1-(三甲氧基甲硅烷基)-1-丙胺(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N,N-二甲基-4-(三乙氧基甲硅烷基)-1-萘胺(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);9-[2-(三乙氧基甲硅烷基)乙基]-9H-咔唑(n=1,A是含咔唑基部分,Z1、Z2和Z3是C1-C4烷氧基);N1,N1,N2-三甲基-N2-[3-(三甲氧基甲硅烷基)丙基]-1,2-乙二胺(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);(氨基乙基氨基甲基)苯乙基三甲氧基硅烷(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);1,3,5-三[3-(三甲氧基甲硅烷基)丙基]-1,3,5-三嗪-2,4,6(1H,3H,5H)-三酮(n=3,A是含异氰脲酸酯部分,Z1、Z2和Z3是C1-C4烷氧基);3-(三乙氧基甲硅烷基)-N-[3-(三乙氧基甲硅烷基)丙基]-1-丙胺(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N1,N1-双[3-(二甲氧基甲基甲硅烷基)丙基]-1,2-乙二胺(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N1,N2-双[3-(三甲氧基甲硅烷基)丙基]-1,2-乙二胺(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);3,3,15,15-四甲氧基-2,7,16-三氧杂-11-氮杂-3,15-二硅十七烷-9-醇(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);3-(间氨基苯氧基)丙基三甲氧基硅烷(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);3-(4-氨基脲基)丙基三乙氧基硅烷(n=1,A是含氨基脲基部分,Z1、Z2和Z3是C1-C4烷氧基);(17-氨基十七烷基)三甲氧基硅烷(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);16-氨基十六烷基三甲氧基硅烷(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N1-[12-(三乙氧基甲硅烷基)十二烷基]-1,2-乙二胺(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N1,N7-双[9-(三乙氧基甲硅烷基)壬基]-1,7-庚二胺(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N1,N1-双[7-(三甲氧基甲硅烷基)庚基]-1,7-庚烷二胺(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N1,N10-双[6-(二甲氧基甲基甲硅烷基)己基]-N1,N10-二甲基-1,10-癸二胺(n=2,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N2-[3-(三甲氧基甲硅烷基)丙基]-N1,N1-双[2-[[3-(三甲氧基甲硅烷基)丙基]氨基]乙基]-1,2-乙二胺(n=3,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N,N,N'-三[3-(三甲氧基甲硅烷基)丙基]-1,2-乙二胺(n=3,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N-[3-(三甲氧基甲硅烷基)丙基]-1-十八胺(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);N-[3-(三甲氧基甲硅烷基)丙基]-1-二十二烷胺(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基);以及N-十八烷基-N'-[3-(三甲氧基甲硅烷基)丙基]-1,3-丙二胺(n=1,A是含胺部分,Z1、Z2和Z3是C1-C4烷氧基)。
另外的可电离改性试剂可以选自以下等等中的一种或多种:3-(二甲氧基苯基甲硅烷基)-1-丙胺;3-氨基丙基二异丙基乙氧基硅烷;N-环己基氨基甲基三乙氧基硅烷;2-(4-吡啶基乙基)三乙氧基硅烷;(N,N-二乙基氨基丙基)三甲氧基硅烷;(3-氨基丙基)三乙氧基硅烷;N-3-[(氨基(聚丙烯氧基)]氨基丙基三甲氧基硅烷;N,N'-双(2-羟乙基)-N,N'-双(三甲氧基甲硅烷基丙基)乙二胺;N-(2-氨基乙基)-3-氨基丙基三甲氧基硅烷;N-环己基-3-氨基丙基三甲氧基硅烷;1-(三甲氧基甲硅烷基)-N-(2-氨基乙基)-3-氨基丙基甲基二甲氧基硅烷3-十八胺;N,N-双[3-(三甲氧基甲硅烷基)丙基]-1-十六胺;以及4,4,7,7-四乙氧基-N-十六烷基-N-丙基3,8-二氧杂-4,7-二硅杂癸烷-5-胺。
还应注意,某些可电离改性试剂除了为色谱表面提供如本文所述的包含一个或多个阴离子交换部分的可电离改性剂之外,还可以同时为色谱表面提供如本文所述的疏水改性剂。此类可电离改性试剂的示例包括N-[3-(三甲氧基甲硅烷基)丙基])-1-十八胺;N-[3-(三甲氧基甲硅烷基)丙基]-1-二十二胺;N-十八烷基-N'-[3-(三甲氧基甲硅烷基)丙基]-1,3-丙二胺;1-(三甲氧基甲硅烷基)-3-十八胺;N,N-双[3-(三甲氧基甲硅烷基)丙基]-1-十六胺;4,4,7,7-四乙氧基-N-十六烷基-N-丙基3,8-二氧杂-4,7-二硅杂癸烷-5-胺。在这些实施方案中,可电离改性剂可以与如本文所述的另外的疏水改性试剂组合使用或者可以独立于这样的另外的疏水改性试剂使用。
在各种实施方案中,在色谱表面已经被提供可电离改性剂和疏水改性剂之后,可以进一步处理该表面以去除任何过量的硅烷醇基团,并通过使该表面与硅烷封端试剂反应来为表面提供额外的有机特征。此类硅烷封端试剂的示例包括一种或多种式SiZ1Z2Z3Z4的硅烷化合物,其中Z1选自Cl、Br、I、C1-C4烷氧基和C1-C4烷基氨基,并且其中Z2、Z3、Z4独立地选自C1-C2烷基。此类硅烷封端试剂的具体示例包括三乙基氯硅烷、三甲基氯硅烷、(N,N二甲基氨基)三乙基硅烷和(N,N二甲基氨基)三甲基硅烷等。
在某些方面,本公开的高纯度色谱材料可以是用于开管柱色谱的颗粒、整料、表面多孔材料、表面多孔颗粒、表面多孔整料或表面多孔层等其他可能形式的形式。
当高纯度色谱材料是颗粒的形式时,高纯度色谱材料的平均粒度可以为约0.3-100μm;约0.5μm-20μm;0.8μm-10μm;或约1.0μm-5.0μm。
在某些方面,本公开的高纯度色谱材料可以是具有芯材料的整料或具有芯材料的颗粒的形式。芯材料可以选自例如有机材料、无机材料或有机-无机杂化材料。
在某些方面,本公开提供了含有高纯度色谱材料诸如上文所描述的那些的色谱分离装置。此类装置的示例包括例如色谱柱、薄层板、过滤膜、微流体分离装置、样品净化装置、固体支持物、固相萃取装置、微芯片分离装置和微量滴定板。
在某些方面,本公开提供了含有高纯度色谱材料诸如上文所描述的那些的色谱柱,其中该柱的内表面由惰性材料形成。
在一些实施方案中,柱可以是由惰性材料形成的非金属柱,例如,聚合物材料,诸如PEEK或PTFE或PFA。
在一些实施方案中,柱可以是涂覆有惰性材料的金属柱。
此类惰性涂覆材料的示例可以选自聚合物涂层、烷基甲硅烷基涂层和源自化学气相沉积的无机涂层。聚合物涂层可以例如由聚醚醚酮(PEEK)形成。烷基甲硅烷基涂层包括具有式II的烷基甲硅烷基涂层,其中R1、R2、R3、R4、R5和R6各自独立地选自(C1-C6)烷氧基、—NH(C1-C6)烷基、—N((C1-C6)烷基)2、OH、ORA和卤基,其中RA表示与柱的内表面的连接点,并且R1、R2、R3、R4、R5和R6中的至少一个是ORA,并且其中X是(C1-C20)烷基、—O[(CH2)2O]1-20—、—(C1-C10)[NH(CO)NH(C1-C10)]1-20-或—(C1-C10)[烷基苯基(C1-C10)烷基]1-20-。这些和其他材料描述于美国专利公开号20190086371中。
在某些方面,本公开提供了采用高纯度色谱材料诸如上文所描述的那些的方法。
在某些方面,本公开提供了一种用于混合模式阴离子交换反相液相色谱的方法,该方法包括:(a)将包含多种酸性分析物的样品装载到包含高纯度色谱材料如上文所描述的那些的色谱分离装置上,使得酸性分析物吸附到高纯度色谱材料上;以及(b)用包含水、有机溶剂和有机酸盐的流动相从高纯度色谱材料洗脱所吸附的酸性分析物,从而分离酸性分析物,其中用流动相从色谱材料洗脱酸性分析物包括这样的洗脱过程,其中流动相的pH随时间推移而改变,流动相的离子强度随时间推移而改变,并且有机溶剂的浓度随时间推移而改变。
在各种实施方案中,样品流体是或来源于生物样品。示例性生物样品包生物流体(例如,全血样品、血浆样品、血清样品、口腔流体、尿液等)、生物组织、生物基质、细胞(例如,一种或多种类型的细胞)、细胞培养上清液、食品(例如,肉类、全谷类、豆类、蛋等)和食品提取物。
在一些实施方案中,酸性分析物是酸性糖类。
在一些实施方案中,酸性分析物是酸性聚糖。酸性聚糖可以包括唾液酸化聚糖、磷酸化聚糖和硫酸化聚糖。此类聚糖物质可以以释放和标记的聚糖、未标记的天然聚糖、未标记的还原聚糖或糖肽的形式进行分析。
在一些实施方案中,有机溶剂包括甲醇、乙醇、1-丙醇、2-丙醇、乙腈、丙酮、乙酸乙酯、甲基乙基酮和四氢呋喃中的一种或多种。
在一些实施方案中,有机酸盐是挥发性有机酸盐,该挥发性有机酸盐包含有机酸阴离子和选自铵阳离子或胺阳离子的阳离子。有机酸阴离子的示例包括例如甲酸根、二氟乙酸根、三氟乙酸根、乙酸根、丙酸根、丁酸根、草酸根、丙二酸根、琥珀酸根、马来酸根、戊二酸根、乙醇酸根、乳酸根、酒石酸根、苹果酸根、柠檬酸根和葡糖酸根等。
在一些实施方案中,流动相还包含有机酸。有机酸的示例包括甲酸、二氟乙酸、三氟乙酸、乙酸、丙酸、丁酸、草酸、丙二酸、琥珀酸、马来酸、戊二酸和有机羟基酸诸如乙醇酸、乳酸、酒石酸、苹果酸、柠檬酸和葡糖酸等等。
在具体实施方案中,流动相包含甲酸和甲酸铵。
在各种实施方案中,用流动相从色谱材料洗脱酸性分析物包括这样的洗脱过程,其中流动相的pH随时间推移而增加,其中流动相的离子强度随时间推移而增加,并且其中并且有机溶剂的浓度随时间推移而增加。
在一些实施方案中,流动相的pH在洗脱过程中增加至少3至10的范围,在一些实施方案中,至少2至12。
在一些实施方案中,流动相的pH在洗脱过程中增加至少2个单位,在一些实施方案中,增加至少4个单位、至少6个单位、至少8个单位、至少10个单位或更多。在一些实施方案中,可以选择某些溶剂梯度以特异性影响缓冲试剂的活性系数和有效pKa。
在一些实施方案中,流动相的离子强度在洗脱过程中从3mmol增加到20mmol。在其他实施方案中,流动相的离子强度可以从有效的零变化到200mmol。
在一些实施方案中,混合至少两种溶液以形成流动相。在一些实施方案中,混合两种溶液以形成流动相,该两种溶液中的第一种溶液是水,并且该两种溶液中的第二种溶液是包含水、有机溶剂、有机酸盐和任选的有机酸的溶液。
在一些实施方案中,该方法还包括使包含多种酸性分析物的样品与标记试剂反应以产生标记的分析物样品,将该标记的分析物样品装载到色谱分离装置上,以及用流动相从高纯度色谱材料洗脱吸附的标记的分析物。
标记试剂可以选自MS活性荧光标记,其示例包括普鲁卡因胺试剂或普鲁卡因试剂。标记试剂可以是Log P值介于0和5之间、通常介于1和5之间、更通常介于1和3之间的两亲强碱性部分。标记试剂可以是具有大于6的pKa值、通常具有大于7的pKa值、更通常具有大于8的pKa值的两亲强碱性部分。
在具体实施方案中,标记试剂是具有下式的试剂:
在其他实施方案中,聚糖标记试剂可以是2-AA(邻氨基苯甲酸)、3-ASA(苯胺磺酸)、APTS(8-氨基芘-1,3,6-三磺酸)、Gly-Q(Prozyme,San Leandro,CA)或其衍生物和异构体。
在一些实施方案中,该方法还包括将来自液相色谱的洗脱液进行质谱分析(MS),其示例包括串联质谱(MS/MS)、电喷雾电离质谱(ESI-MS)、基质辅助激光解吸/电离质谱(MALDI-MS)和飞行时间质谱(TOFMS)。
附图说明
具体实施方式
在具体实施方案中,本公开提供了一种用于混合模式阴离子交换反相液相色谱和酸性聚糖物质(包括但不限于唾液酸化、磷酸化和硫酸化聚糖)的选择性保留的方法。这些聚糖物质可以以释放和标记的衍生物、未标记的天然聚糖、未标记的还原聚糖或糖肽的形式进行分析。该方法有利地将挥发性流动相与增加浓度梯度的铵盐和有机溶剂以及填充有高纯度色谱材料的色谱柱组合,所述色谱柱被设计成最大限度地减少电喷雾电离过程中的干扰。
本公开强调了用具有两个、三个或更多个甲硅烷基的可电离改性试剂(包括双和三甲硅烷基可电离改性试剂)制备的材料的效用。
通过选择合适的可电离改性剂,可以产生非常适合分离用MS增强标记衍生的酸性聚糖的表面化学物质,包括但不限于两亲强碱性部分,诸如Waters RapiFluor-Prozyme Instant Procaine(InstantPCTM)、普鲁卡因和普鲁卡因胺。本文所述的可电离改性剂独特地提供了深深嵌入的可电离表面残基,并且因此显示出增强的色谱选择性。由于这些可电离改性剂具有与其底物的多个连接点,因此它们相应的高纯度色谱材料表现出相对较低水平的所谓色谱柱流失,这有益于在线质谱检测。
在各种优选实施方案中,本公开提供了一种用于聚糖分析的LC-MS方法,该LC-MS方法实现了具有高灵敏度检测的增强的色谱分辨能力,如通过改进的色谱材料实现的,所述改进的色谱材料在与质谱检测一起使用时提供低水平的干扰。
实施例1.
BEH多孔杂化颗粒(按照如美国专利6,686,035中所述的方法制备)完全分散于甲苯(5mL/g)中,然后共沸汽提(回流,1小时)以确保无水条件。将如表1所示的可电离改性试剂S1-S10(0.1-0.3μmol/m2)添加到BEH/甲苯浆液中,然后在室温处搅拌1小时。
表1
然后将浆液在惰性气氛下回流搅拌2小时。然后使反应冷却至室温,并经由过滤分离颗粒。将颗粒依次用甲苯、丙酮/水(1:1v/v)和丙酮洗涤。然后将分离的颗粒在80℃处在25mm真空下干燥16小时。干燥后,将颗粒完全分散于甲苯(10mL/g)中,然后共沸汽提(回流,3小时)以确保无水条件。将碱催化剂(吡啶或咪唑:3.2μmol/m2)添加到颗粒/甲苯浆液中,然后将疏水改性剂(十八烷基三氯硅烷(tC18):1.6μmol/m2)添加到颗粒/甲苯浆液中。将浆液在惰性气氛下回流搅拌20小时。使反应冷却至室温,并经由过滤分离颗粒。将颗粒依次用甲苯、丙酮、丙酮/水(1:1v/v)和丙酮洗涤。将丙酮润湿的颗粒转移到干净的反应器中,分散于丙酮/0.12M乙酸铵(8.2:1.8v/v)中并水解(59℃,2小时)。然后通过过滤分离颗粒并依次用甲苯、丙酮、丙酮/水(1:1v/v)和丙酮洗涤。最后,将分离的表面改性颗粒在70℃处在25mm真空下干燥16小时并进行表征。结果示于表2中。
表2.
可电离改性剂的表面覆盖率通过元素分析测得的表面改性后的颗粒%N的差值来确定。C18疏水改性剂的表面覆盖率通过如元素分析测得的表面改性前后的颗粒%C的差值来确定。%N值通过燃烧分析(CE-440元素分析仪;Exeter Analytical Inc.,NorthChelmsford,MA)来测量,或者%C通过库仑碳分析仪(模块CM5300,CM5014,UIC Inc.,Joliet,Il)来测量。
实施例2.
使得自实施例1的表面改性的颗粒完全分散于甲苯(10mL/g)中,然后共沸汽提(回流,1小时)以确保无水条件。将三乙基氯硅烷(TES)或(N,N-二甲基氨基)三乙基硅烷(TES:5μmol/m2)添加到表面改性的颗粒/甲苯浆液中,然后在惰性气氛下回流搅拌4小时。然后将反应物冷却至室温并将三甲基氯硅烷或(N,N-二甲基氨基)三甲基硅烷(TMS:8μmol/m2)添加到表面改性的颗粒/甲苯浆液中,然后在惰性气氛下回流搅拌16小时。如果使用氯硅烷,则将碱催化剂(吡啶或咪唑:3.2μmol/m2)添加到反应浆液中。将反应物冷却至室温,随后通过过滤分离颗粒并依次用甲苯、丙酮、丙酮/水(1:1v/v)和丙酮洗涤。最终,在25mm真空下,分离的表面改性颗粒于70℃干燥16小时。对颗粒进行了表征,结果呈现于表3中。
表3.
产物 | 颗粒(g) | 前体 | TES质量(g) | TMS质量(g) | 最终碳(%C) |
2a | 8 | 1a | 1.2 | 1.3 | 14.40 |
2b | 8 | 1b | 1.1 | 1.3 | 14.47 |
2c | 8 | 1c | 1.1 | 1.3 | 13.63 |
2d | 8 | 1d | 1.1 | 1.3 | 13.65 |
2e | 8 | 1e | 1.1 | 1.3 | 13.79 |
2f | 8 | 1f | 1.1 | 1.3 | 13.85 |
2g | 19 | 1g | 2.6 | 2.9 | 13.84 |
6h | 8 | 1h | 1.1 | 1.3 | 13.77 |
6i | 8 | 1i | 1.1 | 1.2 | 13.77 |
2j | 8 | 1j | 1.1 | 1.3 | 14.19 |
2k | 8 | 1k | 1.1 | 1.3 | 14.08 |
2l | 8 | 1l | 1.1 | 1.3 | 14.13 |
实施例3.
将如实施例1中所描述的方法扩展为包括0.03-1.0μmol/m2的可电离改性剂覆盖率和2.5:1至67:1的疏水改性剂与可电离改性剂的比率。
实施例4.
将来自实施例3的表面改性的颗粒使用如实施例2中所描述的方法进一步改性。
实施例5.
将如实施例1和3中所描述的方法扩展为包括其他感兴趣的可电离改性剂,例如但不限于表4中的S11-S42与疏水基团的组合以产生2.5:1至67:1的疏水相与可电离改性剂的比率。
表4.
实施例6.
将来自实施例5的表面改性的颗粒使用如实施例2中所描述的方法进一步改性。
实施例7.
将如实施例1的方法扩展为包括其他感兴趣的改性试剂,它们为所得产物提供可电离改性剂和疏水改性剂。此类改性试剂包括但不限于表5中的S43-S48。由于改性试剂包括疏水改性剂,因此可以省去实施例1中的步骤,其中将疏水改性试剂(十八烷基三氯硅烷(tC18)添加到颗粒/甲苯浆液中,回流并冷却。在其他实施方案中,进行实施例1中的步骤,其中将疏水改性试剂(十八烷基三氯硅烷(tC18)添加到颗粒/甲苯浆液中,回流并冷却。
表5.
实施例8.
将来自实施例8的表面改性的颗粒使用如实施例2中所描述的方法进一步改性。
实施例9.
唾液酸化聚糖的阴离子交换/反相液相色谱(RPLC)。已经用根据本公开的材料实现了唾液酸化聚糖的分离,并且已经发现其表现出非常高的分离度。提供了一个示例性实施方案,其中RapiFluor-MS标记的来自牛胎球蛋白(Sigma)的聚糖已经用双甲酸铵/乙腈梯度分离,使用填充有1.7μm桥接的乙烯有机硅全多孔颗粒的色谱柱,所述全多孔颗粒用含双甲硅烷基叔胺的可电离改性剂以及如上所述的三官能键合的C18进行改性。
这种分离的色谱图示于图1中。已经使用牛津符号进行了聚糖分配。例如,A3G3S3表示由三个半乳糖残基(G3)和三个唾液酸残基(S3)改性的三触角核心(A3)组成的聚糖。一些注释包括带有NGNA的括号标记,以表示唾液酸残基以N-乙二醇与N-乙酰基部分的形式存在的位置。在这一点上,可以看出,可以从牛胎球蛋白中解析异构体和聚糖电荷异质性,如通过许多基线解析峰及其注释所例示的。进而,这些结果表明,本公开内容及其在唾液酸化糖蛋白的结构表征中的应用前景广阔,包括从血液因子到酶替代疗法的重组蛋白治疗剂,其通常被设计成表现出高水平的唾液酸化,以便延长循环半衰期或影响炎症反应。
表6.带有荧光和MS检测的阴离子交换RPLC的条件
实施例10
磷酸化聚糖的阴离子交换RPLC。还已经制备了从重组人β-葡糖苷酸酶(NovusBiologics,6144-GH)释放的聚糖,用RapiFluor-标记并使用填充有1.7μm桥接的乙烯有机硅全多孔颗粒的色谱柱进行色谱分析,所述全多孔颗粒用含双甲硅烷基叔胺的可电离改性剂以及如上所述的三官能键合的C18进行改性。聚糖可以在含有甘露糖-6-磷酸酯(M6P)残基的这种类型的样品中发现。在解决溶酶体贮积症的酶替代疗法的情况下,这种残基的量和相应聚糖结构的相对量至关重要。多种酶已投放市场用于患者治疗,包括重组葡糖苷酸酶、半乳糖苷酶、葡糖苷酶、肝素硫酸酯酶,并且这些中的每一种都必须用M6P进行翻译后修饰,以适当地促进细胞摄取。因此,重要的是存在用于检测和定量含M6P聚糖的稳健测定。
这种分离的色谱图示于图2中。已经根据其组成信息(Hex=己糖,HexNAc=N-乙酰基己糖胺)进行了聚糖分配。M5、M6和M9对应于高甘露糖聚糖。根据牛津符号分配FA1G1。重要的是,含有膦基团的聚糖用“P”表示;单磷酸化的聚糖被分配“-P”,并且双磷酸化的聚糖被分配“-2P”。
通过这些分析结果,在聚糖种类的基于电荷的物质形成之上实现了更高的分离度。也就是说,可以看到N-聚糖被洗脱到不带电荷、单电荷、双电荷和三电荷物质的不同池中。在每个色谱区域内,聚糖异质性通过它们的异构化和带电荷残基的单独组成进一步阐明,例如,一个磷酸化残基和两个唾液酸化残基。因此,在很短的时间内产生了大量的信息,并且可以通过光学检测器或在线质谱检测来检测每种物质。
表7.带有荧光和MS检测的阴离子交换RPLC的条件
Claims (59)
1.一种包括色谱表面的色谱材料,其中所述色谱表面包含疏水改性剂和包含在电离时带正电的一个或多个阴离子交换部分的可电离改性剂。
2.根据权利要求1所述的色谱材料,其中高纯度色谱材料在约3至约10的pH范围内是水解稳定的。
3.根据权利要求1至2中任一项所述的色谱材料,其中所述色谱材料是无机材料、杂化有机/无机材料、具有杂化表面层的无机材料、具有无机表面层的杂化材料或具有不同杂化表面层的杂化材料。
4.根据权利要求1至3中任一项所述的色谱材料,其中所述色谱材料包括选自二氧化硅、氧化铝、二氧化钛、氧化锆以及它们的组合的无机材料。
5.根据权利要求1至4中任一项所述的色谱材料,其中所述高纯度色谱材料包括杂化有机/无机材料,所述杂化有机/无机材料包含≡Si-(CH2)n-Si≡部分和/或≡Si–O–(CH2)mCH3部分,其中n是等于1、2、3或4的整数,并且m是等于0、1、2或3的整数。
6.根据权利要求1至5中任一项所述的色谱材料,其中所述色谱材料能够通过水解缩合以下化合物来形成:(a)式SiZ1Z2Z3Z4的一种或多种硅烷化合物,其中Z1、Z2、Z3和Z4独立地选自Cl、Br、I、C1-C4烷氧基、C1-C4烷基氨基和C1-C4烷基,但是Z1、Z2、Z3和Z4中的至多三个能够是C1-C4烷基;和/或(b)式Si Z1Z2Z3—R—SiZ4Z5Z6的一种或多种化合物,其中Z1、Z2和Z3独立地选自Cl、Br、I、C1-C4烷氧基、C1-C4烷基氨基和C1-C4烷基,但是Z1、Z2和Z3中的至多两个能够是C1-C4烷基,并且其中Z4、Z5和Z6独立地选自Cl、Br、I、C1-C4烷氧基、C1-C4烷基氨基和C1-C4烷基,但是Z4、Z5和Z6中的至多两个能够是C1-C4烷基,并且其中R是有机基团。
7.根据权利要求1至6中任一项所述的色谱材料,其中所述疏水改性剂包含具有4至30个碳原子的烃部分。
8.根据权利要求1至6中任一项所述的色谱材料,其中所述疏水改性剂包含C4-C30脂族部分、C4-C30芳族部分、苯基烷基部分或氟芳族部分。
9.根据权利要求1至6中任一项所述的色谱材料,其中所述疏水改性剂包含一个或多个含有8至18个碳原子的烷基基团。
10.根据权利要求1至9中任一项所述的色谱材料,其中所述疏水改性剂以0.1微摩尔/平方米至5微摩尔/平方米范围内的表面浓度存在。
11.根据权利要求1至10中任一项所述的色谱材料,其中所述可电离改性剂包含pKa范围为4至13的阴离子交换部分。
12.根据权利要求1至11中任一项所述的色谱材料,其中所述可电离改性剂包含阴离子交换部分,所述阴离子交换部分选自含胺部分、含胍部分、含脒部分、含吡啶基部分、含咪唑基部分、含咔唑基部分、含异氰脲酸酯部分和/或含氨基脲基部分。
13.根据权利要求11至12中任一项所述的色谱材料,其中所述阴离子交换部分通过六个或更多个甲硅烷氧基键连接到所述色谱表面。
14.根据权利要求1至11中任一项所述的色谱材料,其中所述可电离改性剂包含阴离子交换部分,所述阴离子交换部分选自含胺部分、含胍部分、含脒部分、含吡啶基部分、含咪唑基部分、含咔唑基部分、含异氰脲酸酯部分和/或含氨基脲基部分,所述阴离子交换部分桥接两个或更多个甲硅烷氧基基团,其中每个甲硅烷氧基基团通过三个甲硅烷氧基键连接到所述色谱表面。
15.根据权利要求1至14中任一项所述的色谱材料,其中疏水改性剂:可电离改性剂的摩尔比为约2:1至约100:1。
16.根据权利要求1至15中任一项所述的色谱材料,其中所述可电离改性剂的表面浓度为约0.01μmol/m2至约1.0μmol/m2。
17.根据权利要求1至16中任一项所述的色谱材料,其中所述色谱表面通过使所述色谱材料与包含(a)疏水部分和(b)一个或多个反应性硅烷基团的反应性疏水改性试剂反应而衍生。
18.根据权利要求18所述的色谱材料,其中所述可电离改性试剂具有式M(SiZ1Z2Z3)n,其中n=1、2、3或更大,M表示疏水部分,并且Z1、Z2和Z3独立地选自Cl、Br、I、C1-C4烷氧基和C1-C4烷基氨基。
19.根据权利要求19所述的色谱材料,其中所述疏水部分包含具有4至30个碳原子的烃部分。
20.根据权利要求19所述的色谱材料,其中所述疏水部分是包含C4-C30脂族部分、C4-C30芳族部分、苯基烷基部分或氟芳族部分的烃部分。
21.根据权利要求1至20中任一项所述的色谱材料,其中所述色谱表面通过使所述色谱材料与包含以下项的可电离改性试剂反应而衍生:(a)选自含胺部分、含胍部分、含脒部分、含吡啶基部分、含咪唑基部分、含咔唑基部分、含异氰脲酸酯部分和/或含氨基脲基部分的一个或多个部分和(b)两个或更多个反应性硅烷基团。
22.根据权利要求21所述的色谱材料,其中所述反应性硅烷基团含有选自Cl、Br、I、C1-C4烷氧基和C1-C4烷基氨基的三个反应性基团。
23.根据权利要求1至20中任一项所述的色谱材料,其中所述色谱表面通过使所述色谱材料与可电离改性试剂反应而衍生,所述可电离改性试剂具有式A(SiZ1Z2Z3)n,其中n=1、2、3、4或更大,其中A表示含胺部分、含胍部分、含脒部分、含吡啶基部分、含咪唑基部分、含咔唑基部分、含异氰脲酸酯部分和/或含氨基脲基部分,其中Z1、Z2和Z3独立地选自Cl、Br、I、C1-C4烷氧基、C1-C4烷基氨基和C1-C4烷基,但是Z1、Z2和Z3中的至多两个能够是C1-C4烷基,并且其中当n=2或更大时,-SiZ1Z2Z3基团中的每一个能够彼此相同,或者-SiZ1Z2Z3基团中的每一个能够彼此不同。
24.根据权利要求23所述的色谱材料,其中Z1、Z2和Z3独立地选自Cl、Br、I、C1-C4烷氧基和C1-C4烷基氨基。
25.根据权利要求21至24中任一项所述的色谱材料,其中所述可电离改性试剂选自双甲硅烷基可电离改性剂和三甲硅烷基可电离改性剂。
26.根据权利要求21至25中任一项所述的色谱材料,其中所述可电离改性试剂还包含疏水部分。
27.根据权利要求17至26中任一项所述的色谱材料,其中所述色谱表面通过使所述色谱材料与硅烷封端试剂反应而进一步衍生。
28.根据权利要求27所述的色谱材料,其中所述硅烷封端试剂是式SiZ1Z2Z3Z4的硅烷化合物,其中Z1选自Cl、Br、I、C1-C4烷氧基和C1-C4烷基氨基,并且其中Z2、Z3、Z4独立地选自C1-C2烷基。
29.根据权利要求1至28中任一项所述的色谱材料,其中所述色谱材料是用于开管柱色谱的颗粒、整料、表面多孔材料、表面多孔颗粒、表面多孔整料或表面多孔层的形式。
31.根据权利要求1至30中任一项所述的色谱材料,其中所述色谱材料是具有芯材料的整料或具有芯材料的颗粒的形式。
32.根据权利要求1至31中任一项所述的色谱材料,其中所述色谱材料是平均粒度为约0.3μm-100μm的颗粒的形式。
33.一种色谱分离装置,所述色谱分离装置包含根据权利要求1至32中任一项所述的色谱材料。
34.根据权利要求33所述的色谱分离装置,其中所述色谱分离装置选自色谱柱、薄层板、过滤膜、微流体分离装置、样品净化装置、固体支持物、固相萃取装置、微芯片分离装置和微量滴定板。
35.根据权利要求34所述的色谱分离装置,其中所述色谱分离装置是色谱柱,并且其中所述柱的内表面由惰性材料形成。
36.根据权利要求35所述的色谱分离装置,其中所述柱是由惰性材料形成的非金属柱。
37.根据权利要求35所述的色谱分离装置,其中所述柱是涂覆有惰性材料的金属柱。
38.一种用于混合模式阴离子交换反相液相色谱的方法,所述方法包括:(a)将包含多种酸性分析物的样品装载到包含选自根据权利要求1至32中任一项所述的色谱材料的色谱分离装置上,使得所述酸性分析物吸附到高纯度色谱材料上,以及(b)用包含水、有机溶剂和有机酸盐的流动相从所述高纯度色谱材料洗脱所吸附的酸性分析物,从而分离所述酸性分析物,其中用所述流动相从所述色谱材料洗脱所述酸性分析物包括这样的洗脱过程,其中所述流动相的pH随时间推移而改变,所述流动相的离子强度随时间推移而改变,并且所述有机溶剂的浓度随时间推移而改变。
39.根据权利要求38所述的方法,其中样品流体是或来源于生物样品。
40.根据权利要求38至39中任一项所述的方法,其中所述酸性分析物是酸性糖类。
41.根据权利要求40所述的方法,其中所述酸性分析物是酸性聚糖。
42.根据权利要求41所述的方法,其中所述酸性聚糖选自唾液酸化聚糖、磷酸化聚糖和硫酸化聚糖中的一种或多种。
43.根据权利要求38至42中任一项所述的方法,其中所述有机溶剂包括甲醇、乙醇、1-丙醇、2-丙醇、乙腈、丙酮、乙酸乙酯、甲基乙基酮和四氢呋喃中的一种或多种。
44.根据权利要求38至43中任一项所述的方法,其中所述有机酸盐是挥发性有机酸盐,所述挥发性有机酸盐包含有机酸阴离子和选自铵阳离子或胺阳离子的阳离子。
45.根据权利要求44所述的方法,其中所述有机酸阴离子选自以下中的一种或多种:甲酸根、二氟乙酸根、三氟乙酸根、乙酸根、丙酸根、丁酸根、草酸根、丙二酸根、琥珀酸根、马来酸根、戊二酸根、乙醇酸根、乳酸根、酒石酸根、苹果酸根、柠檬酸根和葡糖酸根。
46.根据权利要求44至45中任一项所述的方法,其中所述流动相还包含有机酸。
47.根据权利要求44所述的方法,其中所述有机酸选自甲酸、二氟乙酸、三氟乙酸、乙酸、丙酸、丁酸、草酸、丙二酸、琥珀酸、马来酸、戊二酸和有机羟基酸诸如乙醇酸、乳酸、酒石酸、苹果酸、柠檬酸和葡糖酸等等。
48.根据权利要求38至47中任一项所述的方法,所述流动相包含甲酸和甲酸铵。
49.根据权利要求38至48中任一项所述的方法,其中用所述流动相从所述色谱材料洗脱所述酸性分析物包括这样的洗脱过程,其中所述流动相的所述pH随时间推移而增加,其中所述流动相的所述离子强度随时间推移而增加,并且其中并且所述有机溶剂的所述浓度随时间推移而增加。
50.根据权利要求49所述的方法,其中所述流动相的所述pH在所述洗脱过程中增加至少3至10的范围。
51.根据权利要求49至50中任一项所述的方法,其中所述流动相的所述pH在所述洗脱过程中增加至少2个单位。
52.根据权利要求49至51中任一项所述的方法,其中所述流动相的所述离子强度在所述洗脱过程中增加至少3mmol至20mmol的范围。
53.根据权利要求49至52中任一项所述的方法,其中混合至少两种溶液以形成所述流动相。
54.根据权利要求49至52中任一项所述的方法,其中混合两种溶液以形成所述流动相,所述两种溶液中的第一种溶液是水,并且所述两种溶液中的第二种溶液是包含水、有机溶剂、有机酸盐和任选的有机酸的溶液。
55.根据权利要求49至54中任一项所述的方法,还包括使包含所述多种酸性分析物的样品与标记试剂反应以产生标记的分析物样品,将所述标记的分析物样品装载到所述色谱分离装置上,以及用所述流动相从所述高纯度色谱材料洗脱吸附的标记的分析物。
56.根据权利要求55所述的方法,其中所述标记试剂是MS活性荧光标记试剂。
57.根据权利要求56所述的方法,其中所述标记试剂是Log P值介于0和5之间的两亲强碱性部分。
58.根据权利要求56所述的方法,其中所述标记试剂是pKa值大于6的两亲强碱性部分。
59.根据权利要求38至58中任一项所述的方法,还包括将来自所述液相色谱的洗脱液进行质谱分析(MS)。
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US20020070168A1 (en) * | 1999-02-05 | 2002-06-13 | Zhiping Jiang | Porous inorganic/organic hybrid particles for chromatographic separations and process for their preparation |
US20100240915A1 (en) * | 2009-02-02 | 2010-09-23 | Robert Jansen | Pre-Treatment of Crude Alcohol or Furan Feed to a Vapor Permeation Apparatus |
CN104220158A (zh) * | 2012-03-28 | 2014-12-17 | 奇奥凯姆股份公司 | 用于反相色谱的掺杂材料 |
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US20190126241A1 (en) * | 2017-09-26 | 2019-05-02 | Waters Technologies Corporation | High purity chromatographic materials comprising an ionizable modifier for retention of acidic analytes |
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