引起峰拖尾的原因很多，我們需要找到問題的根本原因對症下藥！
———AnalyticalLab 點評
Correcting Peak Tailing Problems in Reversed Phase HPLC
反相色譜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中的峰拖尾一直以來都是常見的問題。分離鹼性化合物時峰拖尾尤其普遍，也因此，對於那些通過HPLC分析藥類化合物的人來說，問題始終存在。峰拖尾會導致許多問題，包括分離度降低，靈敏度降低以及精密度和準確度變差。圖1說明了峰拖尾對峰分離度和樣品靈敏度的影響。圖2說明了由於數據系統無法準確識別拖尾峰起點和終點，分析的準確性和精密度會受到影響。
圖1-峰拖尾對分離度和靈敏度的影響
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.
當峰的拖尾因子（T，拖尾因子）從1.0增加到2.0，分離度（Rs）從1.5降低到1.0。當進樣量增加且樣品濃度降低時，靈敏度（峰高）也隨著峰拖尾而降低。
圖2-峰拖尾對準確度和精密度的不利影響
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.
數據系統比較難確定拖尾峰結束的確切位置。因此，準確度和精密度可能會受到影響。在這個例子中，在B點測得的峰面積比在A點測得的峰面積小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,
• 進樣所用溶劑比流動相強的多
• sample mass overload,
• 樣品過載
• stationary phase silianol interactions with amines,
• 固定相中的矽烷醇與胺類相互作用
• adsorption of acidic compounds onto silica, and
• 酸性化合物在矽膠上的吸附
• a void in the column’s packing bed.
• 色譜柱填料床中的空隙。
Once you have identified the cause of the tailing, you can takeaction to reduce or eliminate it. (See Table 1)
一旦你確定了拖尾的原因，你就可以採取措施來減少或消除拖尾（見表1）。
表1-峰拖尾：成因及解決辦法
Peak Tailing Caused by Injecting Sample in a Solvent that is Significantly Stronger than the Mobile Phase
由於進樣溶劑強於流動相而引起的峰拖尾
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.
如果樣品溶於比流動相更強的溶劑中，可能會出現寬而均勻的拖尾峰。有幾條線索可以幫助您確定是否是峰拖尾的原因。第一條線索是，先洗脫的峰後洗脫的峰拖尾更嚴重。另一個線索是，如果注入的樣品量較少或樣品用流動相稀釋，峰拖尾會有所改善。如果您懷疑流動相強度是造成拖尾高峰的原因，則改善方法相當簡單。要麼將樣品溶解在流動相中，要麼用流動相稀釋樣品直到峰拖尾達到可接受的程度。
Peak Tailing Caused by Sample Mass Overload
樣品量過載引起的峰拖尾
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)
當樣品注入量開始超過色譜柱填充容量時，峰的外觀將呈現直角三角形。隨著更多的樣品被注入，峰的前端將變得更尖銳，而峰的後端將更多地拖尾。另一個提示色譜柱過載的線索是，隨著更多樣品質量的注入，保留時間會減少。（見圖3）
圖3-樣品量過載引起的峰拖尾
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.
隨著樣品過載的發生，峰的前端將變得更尖銳，背面峰會尾隨，保留時間會稍微減少。如果峰形看起來有點像一個直角三角形，樣本超載可能發生。
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.
改善由樣品質量超載導致的峰拖尾的方法是注入更少的樣品。表2列出了色譜柱直徑以及在樣品超載出現問題之前建議注入的最大樣品量。表2提供了一系列樣品量，因為實際進樣量取決於色譜柱填料（具有較高表面積的填料可以容納較高的樣品加載量），分析物（較大分子具有較低的加樣量）以及其他因素，如樣品在流動相中的溶解性。
表2-建議注入的最大樣品量
Peak Tailing Caused by Stationary Phase Silanol Interactions with Amines
固定相矽醇基與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中峰拖尾的一個常見原因是在矽膠固定相載體顆粒表面帶正電的溶質（胺）和酸性矽烷醇之間發生離子交換相互作用時發生的二次保留（圖4）。當使用具有顯著矽烷醇活性的固定相的HPLC柱時，通常會遇到這種情況。在中性pH（6至8）下通常比酸性pH（<3）更差。酸性或中性化合物不受影響，一些鹼性化合物比其他化合物更易受影響。
圖4 -峰拖尾的相互作用
Acidic silanols on the surface of silica stationary phase supports can form ion-exchange sites that interact with basic compounds. This ion-exchange interaction will often contribute to peak retention (secondary retention) and cause peak tailing when separating amines by reversed phase HPLC.
二氧化矽固定相載體表面上的酸性矽烷醇基可與鹼性化合物相互作用形成離子交換位點。這種離子交換相互作用通常會促進峰保留（二次保留），在通過反相HPLC分離胺時則會引起峰拖尾。
If you think that silanol interactionis the cause of your peak tailing, there are several steps you can take to correct the problem. First, make sure that the mobile phase is properly buffered (see Table 3) and operate at a pH below 3, if possible. Using sufficient buffer controls the pH and reduces ion-exchange interactions. Operatingat a pH below 3 protonates silanol groups on the silica stationary phase support (pKa of silanol is ~ 3.5) and there by makes the silanols less available for interacting with solutes.
如果您認為矽烷醇相互作用是導致峰拖尾的原因，您可以採取以下幾個步驟來解決問題。首先，如果可以的話，確保流動相經過適當緩衝（見表3）並在pH值低於3的條件下運行。使用足夠的緩衝鹽控制pH並降低離子交換相互作用。在低於3的pH下操作時，使矽膠固定相上的矽烷醇基團質子化（矽烷醇的pKa為約3.5），從而使得矽烷醇與溶質相互作用的可用性降低。
表3-反相高效液相色譜常用緩衝鹽
Another solution to peak tailing is to add a competing amine to the mobile phase. Triethylamine (TEA) is commonly added to mobile phases for this purpose. TEA interacts strongly with silanols and inhibits them from interacting with amines in your sample. Figure 5 is an example of how TEA improves peak shape. About 10 mM TEA is sufficient for most applications.
峰拖尾的另一個解決方法是在流動相中添加競爭胺。為此通常在流動相中添加三乙胺（TEA）。 TEA與矽醇基發生強烈相互作用並抑制它們與樣品中的胺相互作用。圖5是TEA如何改善峰形的一個例子。在大多數實際應用中，大約10mM就足夠了。
Some chromatographers object to adding a competing amine to their mobile phase because it adds complexity to their method and alters the HPLC column in a way that is not easily reversed. Strong amines, such as TEA, are difficult to wash off the column. This means that a column thus modified by TEA is not suitable for applications that do not use TEA in the mobile phase.
有些色譜工作者反對在其流動相中添加競爭胺，因為會使方法更複雜，並會以不可逆的方式改變HPLC色譜柱。強胺如TEA很難從柱子上洗掉。這意味著被TEA改變的色譜柱不適用於流動相中不使用TEA的應用。
圖5-在流動相中加入TEA通常能改善峰形
By adding a competing amine, such as triethylamine (TEA), to the mobile phase, peak tailing can be reduced. TEA interacts strongly with silanols and reduces their ability to interact with amines in your sample mix.
通過在流動相中添加競爭胺如三乙胺（TEA），可以減少峰拖尾。TEA與矽烷醇發生強烈的相互作用，並降低它們與樣品混合物中胺相互作用的能力。
The use of TEA can often be avoided by selecting stationary phases that have very low silanol activity. Figure 6 ranks some popular C18 reversed phase columns according to silanol activity. The ranking in Figure 6 was obtained by measuring the asymmetry of amitriptyline, an amine that is commonly used to measure silanol activityof stationary phases. The less tailing (lower asymmetry value) exhibited by astationary phase when running amitriptyline, the less silanol activity that stationary phase exhibits and the less peak tailing it will have when separating other basic compounds.
選擇具有極低矽烷醇活性的固定相通常可以避免使用TEA。 圖6是根據矽烷醇活性對一些流行的C18反相柱進行的排名。 圖6中的排名是通過測量阿米替林的不對稱性得出的，阿米替林是一種常用於測量固定相矽烷醇活性的胺。 測定阿米替林時呈現的拖尾越小（不對稱值越低），固定相表現出的矽烷醇活性越小，分離其他鹼性化合物時的拖尾峰越少。
圖6-根據矽烷醇活性對一些流行的C18反相柱進行的排名
Amitriptyline is acommonly used probe to test silanol activity of reversed phase HPLC columns. Columns are ranked in this Table according to asymmetry (peak tailing) for amitriptyline. Those with the lowest asymmetry for amitriptyline have the lowest silanol activity. These HPLC columns are usually your best choice when separating amines by reversed phase HPLC.
阿米替林是用於測試反相HPLC柱的矽烷醇活性的常用探針。根據阿米替林的不對稱性（峰拖尾）將色譜柱排列在該表中。阿米替林不對稱性最低的那些具有最低的矽烷醇基活性。當用反相HPLC分離胺時，這些HPLC色譜柱通常是您的最佳選擇。
Data obtained from the National Institutes of Standards & Technology (NIST) Certificate of Analysis for standard reference material 870, 「Column Performance Test Mixturefor Liquid Chromatography.」
數據來自國家標準與技術研究院（NIST）標準參考物質分析證書870，「液相色譜柱性能測試混合物」。
Peak Tailing Caused by Adsorption of Acidic Compounds onto Silica
酸性化合物吸附在矽膠上引起的峰拖尾
Although much less common than peak tailing of amines, acids can sometime show peak broadening and peak tailing because of adsorption onto the silica stationary phase support. To correct peak tailing in these cases increase the salt concentration of the mobile phase to suppress secondary interactions, reducethe mobile phase pH to protonate silanols and solutes and, if necessary, add a competing acid to the mobile phase (Figure 7).
酸儘管比胺的峰拖尾少得多，但由於會吸附在矽膠固定相載體上，有時可能會顯示峰展寬和峰拖尾。為了消除這些情況下的峰拖尾，增加流動相的鹽濃度以抑制二次相互作用，減少流動相pH以使矽醇基和溶質質子化，並且如果需要，向流動相中添加競爭酸（圖7）。
圖7-酸性化合物的峰拖尾通常可以通過在流動相中添加酸來校正
Ibuprofen, an acidic compound, tails badly until acetic acid is added to the mobile phase. The acetic acid preferentially interacts with acidic silanol groups on the surface of the silica stationary phase support and inhibits the interaction between Ibuprofenand silanols that causes peak tailing.
布洛芬是一種酸性化合物，如果不在流動相中加醋酸就會有嚴重的峰拖尾。乙酸優先與矽膠固定相載體表面上的酸性矽烷醇基團相互作用，並抑制引起峰拖尾的布洛芬和矽烷醇之間的相互作用。
Peak Tailing Caused by a Void in the Column’s Packing Bed
柱床填料中的空隙引起的峰拖尾
A void atthe head of the HPLC column’s packing bed will cause peaks to be broad and, sometimes, tail. Although column voids were common several years ago, manufacturers today have perfected their packing techniques and the better quality columns seldom experience a void unless there is a contributing cause, such as operatingat extremely high pressures and/or extremely high flow rates. If, however, you normally get good performance from your HPLC column and then suddenly you start to see tailing on all of your peaks, you may have a column void. The early eluting peaks will be more affected than late eluting peaks by a column void. Although some chromatographers will attempt to repair a column by filling-in the void with a similar stationary phase material, it has been our experience that this is seldom worth the effort. The best cure for a column that is giving tailing peaks because of avoid in the packing bed, is to replace it. This saves time, money, and frustration.
HPLC色譜柱填料床頂部的空隙會導致峰變寬，有時還會形成拖尾。幾年前柱填料空隙還是常見的，但是現在製造商們已經完善了他們的填裝技術，柱體質量更好很少有空隙，除非有某個因素促使空隙發生，例如在極高的壓力和/或極高的流速下運行。但是，如果您的HPLC色譜柱性能一直良好，然後突然發現所有色譜峰都出現了拖尾現象，則可能是色譜柱填料間出現了空隙。先洗脫的峰將比後洗脫的峰更受柱填料空隙的影響。儘管一些色譜工作者會嘗試通過用類似的固定相材料填充空隙來再生色譜柱，但我們的經驗是，這樣做不合算。由於填料床中存在空隙而造成峰拖尾的色譜柱，最好是替換了它。這節省了時間和金錢，減少了挫折。
本篇文章的內容由本公眾號翻譯整理，僅用於學習交流，未能盡善盡美，還請諒解。如有中文意思表達不清楚或錯誤處，請閱讀英語內容。轉載請註明出處~~~
———AnalyticalLab
翻譯：嘻哈小7
校對：Jacopo
排版：Jacopo