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首页 >  资讯  >  技术干货  >  反相色谱峰形拖尾的原因和改善方法

反相色谱峰形拖尾的原因和改善方法

2018-05-16 11:04:09  来源: 检测家

Correcting Peak Tailing Problems in Reversed Phase HPLC

反相色谱峰拖尾的原因和改善方法


Peak tailing in reversed phase HPLC continues to be a common complaint. It is particularly prevalent when separating basic compounds and, therefore, a source of constant problems to those analyzing pharmaceutical compounds by HPLC. Peak tailing causes a number of problems, including lower resolution, reduced sensitivity, and poorer precision and quantitation. Figure1 illustrates how resolution between peaks and sample sensitivity is negatively affected by peak tailing. Figure 2 illustrates how accuracy and precision of ananalysis can suffer because of the inability of data systems to identify exactly where a tailing peak begins and ends.

反相HPLC中的峰拖尾一直以来都是常见的问题。分离碱性化合物时峰拖尾尤其普遍,也因此,对于那些通过HPLC分析药类化合物的人来说,问题始终存在。峰拖尾会导致许多问题,包括分离度降低,灵敏度降低以及精密度和准确度变差。图1说明了峰拖尾对峰分离度和样品灵敏度的影响。图2说明了由于数据系统无法准确识别拖尾峰起点和终点,分析的准确性和精密度会受到影响。

1-峰拖尾对分离度和灵敏度的影响

29f1bb97fc8898d47442a573ef260d46.jpg

 As peak tailing (T, Tailing factor) increases from 1.0 to 2.0, resolution (Rs) decreases from 1.5 to 1.0. Sensitivity (peak height) also decreases with peak tailing since the peak volume increases and the sample concentration decreases.

当峰的拖尾因子(T,拖尾因子)从1.0增加到2.0,分离度(Rs)从1.5降低到1.0。当进样量增加且样品浓度降低时,灵敏度(峰高)也随着峰拖尾而降低。

2-峰拖尾对准确度和精密度的不利影响 

6bdb39e9db039a6f74519330cdb6eb03.jpg

Tailing peaks make it more difficult for data systems to identify exactly where a peakends. Because of this, accuracy and precision can suffer. In this example, the peak area measured at point B is 3% less than the peak area measured at point A.

数据系统比较难确定拖尾峰结束的确切位置。因此,准确度和精密度可能会受到影响。在这个例子中,在B点测得的峰面积比在A点测得的峰面积小3%。

Causes of Peak Tailing

峰拖尾的原因

The major causes of peak tailing include:

造成峰拖尾的主要原因有:

• injecting sample in a solvent that is significantly stronger than the mobile phase,

• 进样所用溶剂比流动相强的多

• sample mass overload,

• 样品过载

• stationary phase silianol interactions with amines,

• 固定相中的硅烷醇与胺类相互作用

• adsorption of acidic compounds onto silica, and

• 酸性化合物在硅胶上的吸附

• a void in the column’s packing bed.

• 色谱柱填料床中的空隙。

 

Once you have identified the cause of the tailing, you can takeaction to reduce or eliminate it. (See Table 1)

一旦你确定了拖尾的原因,你就可以采取措施来减少或消除拖尾(见表1)。

1-峰拖尾:成因及解决办法 

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Peak Tailing Caused by Injecting Sample in a Solvent that is Significantly Stronger than the Mobile Phase

由于进样溶剂强于流动相而引起的峰拖尾


If the sample is dissolved in a solvent that is stronger than the mobile phase, broad and even tailing peaks can occur. There are several clues to help you identify if this is the cause of peak tailing. The first clue is that early eluting peaks have more tailing than late eluting peaks. Another clue is that peak tailing improves if less sample volume is injected or if the sample is diluted with the mobile phase. If you suspect that mobile phase strength is the cause of your peak tailing, the cure is fairly simple. Either dissolve your sample in mobile phase, or dilute your sample with mobile phase to the point where the peak tailing is acceptable.

如果样品溶于比流动相更强的溶剂中,可能会出现宽而均匀的拖尾峰。有几条线索可以帮助您确定是否是峰拖尾的原因。第一条线索是,先洗脱的峰后洗脱的峰拖尾更严重。另一个线索是,如果注入的样品量较少或样品用流动相稀释,峰拖尾会有所改善。如果您怀疑流动相强度是造成拖尾高峰的原因,则改善方法相当简单。要么将样品溶解在流动相中,要么用流动相稀释样品直到峰拖尾达到可接受的程度。

 


Peak Tailing Caused by Sample Mass Overload

样品量过载引起的峰拖尾


When the sample mass injected begins to exceed the capacity ofthe column packing, the peaks will take on the look of a right triangle. As more sample mass is injected, the front of the peak will become sharper and the back of the peak will tail more. Another clue that the column is being overloaded is that retention will decrease as greater sample mass is injected. (See Figure 3)

当样品注入量开始超过色谱柱填充容量时,峰的外观将呈现直角三角形。随着更多的样品被注入,峰的前端将变得更尖锐,而峰的后端将更多地拖尾。另一个提示色谱柱过载的线索是,随着更多样品质量的注入,保留时间会减少。(见图3

3-样品量过载引起的峰拖尾

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As sample overload occurs, the front of peaks will become sharper, the back of the peak will tail, and the retention time will decrease slightly. If the peak shape looks somewhat like a right triangle, sample overload is likely occurring.

随着样品过载的发生,峰的前端将变得更尖锐,背面峰会尾随,保留时间会稍微减少。如果峰形看起来有点像一个直角三角形,样本超载可能发生。

 

The cure for peak tailing caused by sample mass overload is to inject less sample. Table 2 provides a list of column diameters and there commended maximum sample mass that can be injected before sample overload appears as a problem. Table 2 provides a range of sample size because the actual amount will depend on the column packing (packing materials with higher surface area have higher sample loading), the analyte (larger molecules have lower loading), and other factors such as sample solubility in the mobile phase.

改善由样品质量超载导致的峰拖尾的方法是注入更少的样品。表2列出了色谱柱直径以及在样品超载出现问题之前建议注入的最大样品量。表2提供了一系列样品量,因为实际进样量取决于色谱柱填料(具有较高表面积的填料可以容纳较高的样品加载量),分析物(较大分子具有较低的加样量)以及其他因素,如样品在流动相中的溶解性。

2-建议注入的最大样品量

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Peak Tailing Caused by Stationary Phase Silanol Interactions with Amines

固定相硅醇基与胺相互作用引起的峰拖尾


A common cause of peak tailing in reversed phase HPLC is the secondary retention that occurs when an ion-exchange interaction takes place between a positively charged solute (amine) and an acidic silanol on the surface of silica stationary phase support particles (Figure 4). It is observed most often when using HPLC columns packed with stationary phases that have significant silanol activity. It usually is worse at neutral pH (6 to 8) than at acidic pH (<3). Acidic or neutral compounds are not affected, and some basic compounds are more adversely affected than others.

反相HPLC中峰拖尾的一个常见原因是在硅胶固定相载体颗粒表面带正电的溶质(胺)和酸性硅烷醇之间发生离子交换相互作用时发生的二次保留(图4)。当使用具有显著硅烷醇活性的固定相的HPLC柱时,通常会遇到这种情况。在中性pH68)下通常比酸性pH<3)更差。酸性或中性化合物不受影响,一些碱性化合物比其他化合物更易受影响


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