《液相故障排除指南(六)：峰拖尾的原因.docx》由会员分享，可在线阅读，更多相关《液相故障排除指南(六)：峰拖尾的原因.docx（4页珍藏版）》请在taowenge.com淘文阁网|工程机械CAD图纸|机械工程制图|CAD装配图下载|SolidWorks_CaTia_CAD_UG_PROE_设计图分享下载上搜索。
1、6. Sources of PeakTailing6.峰拖尾的原因Q.：What can I do to get rid of peak tailing?问：我要怎样做才能防止峰拖尾呢？A.: First, we will have to find out, where the peaktailing comes from. There are many sources of peak-tailing, ranging from columnproblems to chemistry problems to instrument problems. The most common reason
2、sfor peak tailing are extra-column band broadening, deterioration of the packedbed, and interaction of the analytes with active sites on the packing.Obviously, what needs to be done depends on the cause of tailing.答：首先，我们需要找到是什么原因导致的峰拖尾。造成峰拖尾的原因很多，范围从色谱柱 问题到化学问题再到仪器问题都有可能。引起峰拖尾的最常见原因包括额外的柱谱带增宽， 填料床变
3、质以及被分析物与填料上的活性位点相互作用。那么，根据拖尾原因需要做什么就 很明显了。Q.：I accept that. Now, how do I determine the cause oftailing?问：我同意。现在，我应该如何确定拖尾的原因呢？A.: A quick first step is a careful examination of thechromatogram. There is a lot of information in a chromatogram that can give youclues about the nature of the problem wi
4、thout any knowledge about the samplesor the chromatographic conditions. You can then use the additional informationto test the hypotheses that you have formed based on the examination of thechromatogram.答：快速的第一步是仔细的检查色谱图。色谱图中有很多信息，他们能为你提供有关问题性 质的线索，而无需任何有关样品和色谱条件的信息。然后你可以用这些附加的信息去验证这 个基于检查色谱图而形成的假设。
5、One of the first things to examine is the height of thepeaks. If a UV detector has been used and the peak-heights are in the order ofl AUFS, a good guess is that the column is overloaded and that peak-tailing isdue to overload. This judgement supposes that the extinction coefficients forthe compound
6、s is in the order of 1000, which is a reasonable rule of thumb. Toconfirm mass overload, you would then ask the question of how much mass hasbeen injected onto the column. For a normal, fully porous packing with a pore-size of about 100 A, overload starts distorting peaks at a load of about lOOjig.首
7、先需要检查的就是色谱峰的峰高。如果使用的是UV检测器，且峰高在1AUFS左右，那么很 有可能是色谱柱超载，且峰拖尾就是色谱柱超载引起。该判断假设化合物的吸光系数约为 1000,这一假设是根据经验合理推测得到的规律。为了确认是否为质量超载引起，你需要知 道被注入色谱柱的分析物的量有多少。正常情况下，孔径约为100 A的全多空填料，在约 100 ng的载量下开始出现柱超载和峰变形。These are all rules of thumb, but they can be used asreasonable guidelines for sample overload. You can test f
8、or overload by injectingabout 10 x less mass on the column and see, if the peak-shape improves.这些都是根据经验得到的规律，但是它们可以用来对样品超窄进行合理指导。您可以通过在 色谱柱上进样约x/10倍以下的质量来测试过载，并观察峰形是否有所改善。Q.：Ok. But what if tailing occurs at much lower amountsinjected?问：好的。但如果拖尾是发生在进样量很低的情况下呢？A.: Once again, a peek at the peaks in t
9、he chromatogramis helpful. If there are many peaks in the chromatogram, determine whether thepeak-shape remains roughly constant or if there is a consistent change ofpeak-shape throughout the chromatogram. If the peaks are tailing more in theearly part of the chromatogram than in the later part, one
10、 would suspect thatextra-column effects are responsible. The influence of extra-column effectsdecreases as the peaks become wider, which is why they distort early peaks morethan later peaks. Two extra-column effects should be considered:1. extra-columnband broadeningdetectortime-constant.答：再说一次，看看色谱
11、图中的峰会很有帮助。如果色谱图中有很多峰，判断一下峰形是否 保持大致恒定或整个色谱图中的峰形是否出现了一样的变化。如果色谱图中保存时间靠前的 峰比保存时间靠后的峰拖尾更多，那么可以猜想柱外效应是造成这种情况的原因。柱外效应的 影响会随着峰变宽而减小，这就是为什么它们保存时间靠前的峰比保存时间靠后的峰变形更 大的原因。应考虑以下两个柱外效应：1 . 柱外谱带展宽.检测器的时间常数Band spreading in connection tubing, injectors and detectorsresults in tailing in the early peaks in the chrom
12、atogram. You may encounterthis, if you put a column with a small diameter on an instrument that was notconfigured for the use of small volume columns, or if you have recentlyre-plumbed your system. If the latter is the case, examine the type of tubingthat you have used (it should be 9/1000 i.d. thro
13、ughout from injector todetector). Also inspect all the connections, if they were made properly. Acommon cause of extra-column band spreading is the fact that different columnmanufacturers use different distances between the tip of the ferrule and theend of the tubing in their column end-fittings. An
14、 extra-column band spreadingwith a standard deviation of only 15 |iL significantly influences peak shape upto an elution volume of about 3 ml on a 5 im column.连接管，进样器和检测器中的谱带扩散会导致色谱图中保存时间靠前的峰出现拖尾。如果您 在未配置使用小体积色谱柱的仪器上安装了小直径色谱柱，或者最近重新放置了系统，那么可 能会遇到这种情况。如果是后者，请检查您使用的管子的类型(从进样器到检测器间的应为 9/1000 “ id)。还需要检
15、查所有的接头是否连接正确。柱外谱带展宽的一个常见原因就是 不同的色谱柱制造商在其色谱柱末端配件中使用的密封垫圈尖端到管路末端之间的距离不 同。仅仅15同标准偏差的柱外谱带展宽就会严重的影响到峰形，其影响程度相当于在5 色谱柱上约3 ml的洗脱体积。A large detector time constant has the same influence.More tailing is observed on early peaks than on late eluting peaks. Often, thedefault setting of the time constant is 1 se
16、c. If a high-performance 5 |imcolumn is used, a distortion of the peaks can be observed up to 2 to 3 minutesinto the chromatogram. Larger time constants obviously would give largereffects.一个大的检测器时间常数也有相同的影响。与后洗脱峰相比，先被洗脱的峰中可以观察到更 多的拖尾。通常，时间常数的默认设置为1秒。如果使用高性能的5Hm色谱柱，那么在色谱 图中最多2至3分钟内可以观察到峰的变形。显然，时间常数越大
17、，影响也就越大。If the peaks are all tailing pretty much to the samedegree for all sample compounds in the chromatogram, there are twopossibilities:1. the packed bedis damagedall samplecomponents are chemically similar, and we are dealing with chemical effects. 如果色谱图中的所有样品组分都表现出了想通过程度的严重拖尾，那么有以下两种可能： 1. 填充床
18、损坏2.所有样品组分化学性质相似，我们是与化学效应打交道。In the first case, a plate-count test under standardconditions, especially under conditions recommended by the manufacturer, willreveal whether the column has deteriorated or not. The column could have beendamaged by adsorption of contaminants or particles. Sometimes, it
19、 is possibleto remove these contaminants with an appropriate solvent (for example THF on areversed-phase column), but if the bed itself has shifted, there is nothingthat one can do to repair the column.对于第一种情况，在标准条件下，特别是在色谱柱生产商建议的条件下进行的理论板数测试 将揭示色谱柱是否已损坏。色谱柱可能被吸附的污染物或微粒损伤。有时，可以用适当的溶 剂(例如反相色谱柱上的THF)除
20、去这些污染物，但是如果柱床本身已变性，那么无法进行任 何修复。In the case that all peaks are chemically similar,chemical effects are potential candidates as causes of tailing. If only somepeaks in the chromatogram are tailing and other peaks give a good peak shape,chemical effects are the prime candidates as the causes of peak ta
21、iling.如果是所有峰化学性质类似的情况，化学效应就是引起峰拖尾的潜在因素。如果色谱图中仅 某些峰出现拖尾，而其他峰峰形良好，那么化学效应是导致峰拖尾的主要原因。Q.：What are these chemical effects? Pleaseexplain!问：请解释下，什么是“化学效应”？A.: There could be several effects here as well, but themost common cause is the interaction of the analytes with an energeticallynon-uniform surface.
22、A typical example is the tailing of strong bases on somereversed-phase packings. These kind of compounds interact strongly withresidual silanol groups on the surface of the packing as well as with thebonded phase. If the silanol groups on the surface form a non-homogeneouspopulation, tailing can res
23、ult.答：化学效应也包括好几种，但最常见的是被分析物与能量不均匀外表的相互作用。一个典 型的例子是强碱性化合物在一些反相填料柱上拖尾。这一类化合物会与填料说明残留的硅醇 基和固定相发生强烈的相互作用。如果外表硅醇基分布不均匀，拖尾就发生了。Q.：What can I do to eliminate tailing due to chemicalinfluences?问：我应该如何消除因化学效应引起的拖尾呢？A.: First, you should consider using a column that doesnot exhibit this phenomenon. Some of th
24、e newer reversed-phase packings havebeen designed to minimize the tailing of bases. Second, this tailing can bemediated by either the pH of the mobile phase or by using organic bases in themobile phase that compete with the analytes for active sites. This is due tothe fact that silanophilic interact
25、ions are often ion- exchange interactions.At acidic pH, fewer silanols are negatively charged. Therefore, less tailing isobserved for positively charged analytes. As a competing base, triethylamine isoften used. But among competing bases, those with a larger hydrophobicity oftenwork better. Examples
26、 are octylamine or tetrabutylammonium salts. 答：首先，你应该考虑使用一根不会出现此现象的色谱柱。一些较新的反相填料已被设计用 于最大程度地减少碱基拖尾。其次，这种拖尾可以通过调节流动相pH或向流动相中加入能 与被分析物竞争活性位点的有机碱来减轻。这是因为亲硅醇基相互作用通常是离子交换反响。 在酸性pH条件下，带负电荷的硅醇基减少。因此，对带正电荷的被分析物来说，拖尾现象 就减少了。经常使用三乙胺作为竞争性碱。但是，在竞争碱中，疏水性较大的碱基通常效果 更好。如辛胺或四丁基镂盐。In the case that the silanophilic in
27、teraction of theanalyte are not ion exchange, but hydrogen bonding, you can improve peak-shapeby changing the hydrogen bonding character of your solvent. For example,methanol often results in an improved peak-shape compared to acetonitrile asorganic modifier of the mobile phase.如果被分析物的亲硅醇基相互作用不是离子交换
28、、而是氢键相互作用，你可以通过改变所用 溶剂的氢键键合特性来改善峰形。例如，与乙月青相比，甲醇做流动相的有机改性剂通常峰形 更好。Similar phenomena are encountered in normal phasechromatography, and similar reasoning can be applied there to suppress tailing.Significant changes in tailing can be observed depending on whether alcohols oracetonitrile are used as pol
29、ar modifier of the mobile phase.在正相色谱法中也会遇到类似的现象，并且可以在此采用类似的原理来抑制拖尾。采用乙醇 或乙月青作为流动相的极性改性剂，拖尾情况可得到显着改善。There are still some other causes of peak-tailing, butthey are comparatively rare. Adsorption of sample constituents to column fritsor injector parts has been observed. It is also possible that the analyte issubject to a chemical change during the chromatographic process. Examples ofthis can be a degradation or a slow equilibrium between different molecularforms of the analyte.还有其他一些可能导致峰拖尾的原因，但是都比拟少见。这些原因目前观察到有被吸附到柱 筛板或进样口局部上的样品组分。也有可能是被分析物在色谱别离过程中发生了化学变化。 这样的例子可以是被分析物的降解或是被分析物不同分子形式之间的缓慢的平衡