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色谱峰拖尾怎么办?

分析实验室2020-03-20 10:31:59


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峰拖尾其实并不会直接影响定量,

峰拖尾导致积分不准确,

从而导致定量不准确!


———AnalyticalLab 点评

Reversed-Phase HPLC of Basic Compounds: Solving Peak Tailing Problems

碱性化合物的反相高效液相色谱法:解决峰拖尾问题


What Causes Peak Tailing?

什么会引起峰拖尾?


It is generally believed that an ionexchange interaction between positively charged solutes and acidic silanols on the surface of silica support particles causes peak tailing(Figure 1). Metal impurities on the silica surface can contribute to peak tailing by activating silanol groups.

人们普遍认为,带正电荷的溶质离子和酸性硅羟基在硅胶粒子表面的离子交换作用是造成峰拖尾的原因(图1)。硅胶表面的金属杂质可以激活硅羟基而造成峰拖尾。

 

图1-峰拖尾的相互作用

Acidic silanols on the surface of silica stationary phase supports can form ionexchange sites that interact with basic compounds. This ion-exchange interaction will often contribute to peak retention and cause peak tailing.

酸性硅羟基硅胶固定相载体表面可形成与碱性化合物相互作用的离子交换位点。这种离子交换作用通常会导致峰滞留并引起峰拖尾。 

How Can Peak Tailing Be Corrected?

如何改善峰拖尾?


Peak tailing can be corrected by using mobile phase conditions that reduce the solute/silanol ion-exchange interaction, and by choosing a stationary phase that exhibits less silanol activity.

峰拖尾可以通过选择溶质/硅羟基相互作用更小的流动相,或选择硅羟基活性更小的固定相来改善。

 

Recommended Mobile Phase Conditions for Reducing Peak Tailing

推荐的减少拖尾峰的流动相条件


■Operate at a pH less than 3.0

- Suppresses the ionization of acidic silanols

■在pH小于3.0的流动相条件下操作

-抑制酸性硅羟基的电离

 

■Use a buffer with a concentration between 0.05 and 0.20 M

-Controls pH and reduces ion-exchange interactions

■使用0.05-0.20M之间浓度的缓冲盐

-控制pH值,减少离子交换作用

 

■And, if necessary, add a competing amine, such as 0.01 M triethylamine (TEA)

-Blocks silanol sites from interacting with basic solutes

■必要的时候,添加一个竞争胺,如0.01M三乙胺(TEA)

-阻滞硅羟基团与碱性溶质相互作用

 

These mobile phase conditions will provide acceptable peak symmetry for basic compounds on most reversed phase columns, even those packed with stationary phases that have high silanol activity (Figure 2).However, if you would rather avoid using amine additives in the mobile phase, or if you cannot use a mobile phase pH as low as 3.0 because of compound stability problems or unacceptable resolution at low pH, you should select a stationary phase that has low silanol activity. These types of phases are generally referred to as "base-deactivated" phases (Table 1).

这些流动相条件可以使碱性化合物在使用反相色谱柱洗脱时峰的对称性达到可接受的程度,即便那些色谱柱装填了具有高硅羟基活性的固定相(图2)。然而,如果想避免在流动相中使用胺添加剂,或者由于化合物的稳定性或低pH下的不溶性而不能使用pH值低至3.0的流动相,那么应该选择具有较低硅羟基活性的固定相。这些类型的固定相通常被称为“碱钝化”固定相。(表1)


图2-TEA对峰拖尾的影响

Adding TEA to the mobile phase will usually produce acceptable peak shapes for basic compounds, even on stationary phases with high silanol activity.

添加三乙胺的流动相通常会产生可接受的基本化合物的峰的形状,即使在固定相的硅羟基活性高时。

 

表1-一些常用的C18反相色谱柱,根据硅羟基活性排名

Note: The ranking in this table is meant to provide only a relative comparison of silanol activity. Differences between successive listings may not be significant.

注:本表排名只是提供硅羟基活性的相对比较。列表中连续排名之间并无显著差异。

 

Base-Deactivated Phases Reduce Peak Tailing

碱钝化固定相减少峰拖尾


Base-deactivated phases have fewer acidic silanols available to cause peak tailing.These phases are typically made with low activity silica (high purity "Type B" silica) and have their surface highly covered by the bonded phase. Efficient end-capping with trimethylsilane is one ofthe ways manufacturers use to increase the surface coverage by the bonded phase and further reduce the number of silanols available to cause peak tailing (Figure 3, page 3).

碱钝化固定相有更少的能引起的峰拖尾的酸性硅羟基。这些固定相通常由低活性硅胶(高纯度的“B型”硅胶)制成,其表面被键合相高度覆盖。用三甲基硅烷进行有效的末端封尾是生产上常用的一种钝化方式,可以增加键合相的表面覆盖,进一步减少可造成峰拖尾的硅羟基(图3,第3页)。

 

图3-封尾改善碱性化合物的保留行为

End-capping improves peak shape for basic compounds by further reducing the number of available silanols that can cause peak tailing.

封尾通过显著减少可造成峰拖尾的硅羟基而优化峰形

 

Although most base-deactivated phases will improve peak tailing, they do not eliminate the need for buffering the mobile phase.Column-to-column reproducibility and even injection-to-injection reproducibility can be a problem if the mobile phase is not buffered. In addition, some samples may still require an amine modifier added to the mobile phase to achieve acceptable peakshape. Amine modifiers are more likely to be needed if the mobile phase pH is greater than 4.

虽然大多数碱钝化固定相将改善峰拖尾,但它们并不能消除使用缓冲流动相的必要性。如果流动相没有缓冲能力的话,柱与柱之间的重现性和进样与进样之间的重现性将会是问题。此外,一些样品可能还需要在流动相中加入胺改性剂以达到可接受的峰形。如果流动相pH大于4.0,则可能需要胺改性剂。

 

Select Base-Deactivated Phases that Have the Lowest Silanol Activity for Particularly Difficult Basic Compounds

遇到特别难解决的碱性化合物的时候选择具有最低硅羟基活性的碱钝化固定相


Some basic compounds, such as amitriptyline, will tail even on basedeactivated phases. For these types of difficult basic compounds add an amine modifier to the mobile phase, or select a column packed with a phase that has extremely low silanol activity (Table 1, page 2). With proper mobile phase buffering, these columns can usually provide acceptable peak shape and dependablee producibility without having to add an amine modifier(Figure 4).

一些像阿米替林之类的碱性化合物,在使用碱钝化固定相的时候仍然可能拖尾。对于这类比较难解决的碱性化合物,应该在流动相中添加胺类改性剂,或选择使用极低硅羟基活性填料的色谱柱(表1,第2页)。有了适当的流动相缓冲,用这些色谱柱通常可以得到可接受的峰形和可靠的重复性,而不必添加胺类改性剂(图4)。

 

图4-极低硅羟基活性的色谱柱可以让难解决的碱性化合物得到可接受的峰形

Both Symmetry C18 and Ace C18 are base-deactivated phases with low silanol activity. However, the extremely low silanol activity of Ace C18 provides better peak shape for difficult basic compounds such as amitriptyline.

Symmetry C18和Ace C18都是低硅羟基活性的碱钝化固定相。然而,Ace C18具有更低的硅羟基活性,可以使阿米替林之类的碱性化合物得到更好的峰形。

 

New Ace C18 and C8 Ultra-Inert Base-Deactivated HPLC Columns

Ace®C18C8超惰性碱钝化柱


Ace Stationary Phases Virtually Eliminate the Negative Effects of Silanols on Reversed-phase Separations

Ace固定相几乎可以完全消除硅羟基在反相分离中的负面影响


Ace C18 and C8 columns are manufactured using ultra-pure silica that has extremely low silanol activity. This ultra-pure silica is efficiently bonded andexhaustively end-capped using proprietary technology. The result is a silicabased stationary phase that has virtually eliminated the negative effectsof silanols on reversed-phase HPLC separations.

Ace C18和C8柱均使用具有极低硅羟基活性的超纯硅胶填料。采用专用技术将超纯硅胶彻底封尾并有效地键合,得到的硅胶固定相可以几乎完全消除在反相高效液相色谱法分离过程中硅羟基带来的负面影响。

 

Excellent Peak Shape for Basic and Acidic Compounds

碱性和酸性化合物均能得到极佳峰形


The ultra-inert characteristics of the Ace columns make them the ideal choice for separating polar basic compounds. When compared to other modern base-deactivated columns, the Ace columns consistently produce measurably better peak shape and column efficiency when separating trouble some basic compounds. 

Ace柱的超惰性特征使它们成为分离极性碱性化合物的理想选择。相比其他流行的碱钝化柱,Ace柱在分离较难解决的碱性化合物时可以使峰形更佳,柱效更高。

 

Validated Column-to-Column Reproducibility

经过验证的柱与柱之间的重现性


A series of rigorous quality assurance tests are performed in the manufacturing process to confirm column-to-column reproducibility. This includes three separate chromatographic tests to validate reproducibility on neutral, acidic, and basic compounds.With the tightest specifications in the industry, these columns are guaranteed to provide the best reproducibility of any reversed phase HPLC columns.

在生产过程中会进行一系列严格的质量保证测试,以确认柱与柱之间的重现性。这包括三个独立的色谱试验,以验证中性、酸性和碱性化合物应用中的重现性。按照行业中最严格的规格要求,保证Ace柱在所有反相高效液相色谱柱中具有最好的重现性。

 

图5-阿米替林的色谱洗脱效果

Amitriptyline is commonly used to demonstrate silanol activity on HPLC columns. The Ace columns provide measurably better peak shape and column efficiency compared to other popular base-deactivated columns.

阿米替林通常被用来证明高效液相色谱柱的硅羟基活性。使用Ace柱较其他流行的碱钝化柱明显可以得到更好的峰形和柱效。


本篇文章的内容由本公众号翻译整理,仅用于学习交流,未能尽善尽美,还请谅解。如有中文意思表达不清楚或错误处,请阅读英语内容。转载请注明出处~~~

———AnalyticalLab

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翻译:嘻哈小7

排版:Jacopo


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