液相故障排除指南：峰面积波动.docx

13 .峰面积波动Q.:What are the reasons for fluctuations in peakarea, when the same sample is injected multipletimes?问：同一样品多次注入时，峰面积波动的缘由是什么？A.:Unfortunately, there are many different reasons, too many to count. Nearly every part of the HPLC instrument can conceivably contribute to changes in peak area. Let us review them one by one.答：不幸的是，有很多不同的缘由。HPLC仪器的每个部分都可以导致峰面积的变化。让我 们一一检查。The inject or comes to mind first. What problems you might encounter depends to some degree on the type of injector. But in all cases, the formation of air bubbles during the measurement of the injection volume is a major problem source.If you areinjecting manually,makesure that thereare no air bubbles in your syringe. With fixed-loop injectors,make sure that no air is siphoned into theinjector loop. Inautoinjectors, airbubbles can be formed if the cap of the vial seals well around the needle,which can give rise to a vacuum when a large portion of the sample is injected. Any air bubble form ation results in a random variation of the injection volume from injection to injection.首先想到进样器。您可能遇到的问题在某种程度上取决于进样器的类型。但是在全部状况下, 在测量进样体积期间气泡的形成是主要的问题来源。假如您是手动进样，请确保注射器中没 有气泡。使用固定回路进样器时，确保没有空气进入进样器环路。在自动进样器中，假如样 品瓶的盖子密封在针头四周则会形成气泡，当注入大部分样品时会产生真空。任何气泡的形 成都会导致每次进样之间进样量的差异。sample and the peak areaobtainedfrom a singlesample becomes constantonly after two or three injections,samplecarry-overis a likely source of the problem. Cleanthe syringe carefully after each injection, if you are injectingmanually, or check the needle wash in anauto injector. Makesure that thesolvent used for the needle wash is a goodsolventfortheanalyte(s).假如样品之间分析物的浓度差异很大，并且从单个样品获得的峰面积仅在两次或三次进样后 才保持恒定，样品残留可能是问题的根源。每次注射后(假如您是手动注射)请认真清洗注 射器，或在自动进样器中检查针头清洗。确保用于针头清洗的溶剂是分析物的良好溶剂。Decreasesin peak area from run to run can also be causedby temperature changes, but the effect is small, under 2%. Ifyou take a sample out of the refrigeratorand put it into anautoinjectorjtwillslowlywarmuptotheinjectortemperature and the solvent will expand.Therefore you willinject initially alarger mass of sample than later when thesamplehasreachedthetemperatureofthesamplecompartment.每次运行时峰面积的削减也可能是由于温度变化引起的，但是影响很小，不至IJ 2%。假如将 样品从冰箱中取出并放入自动进样盘中，它将缓慢升温至进样器温度，并且溶剂会膨胀。因 此，当样品达到样品盘的温度时，您开头时注入的样品体积比随后的大。Randomvariationsoftheflowratecanalsocausefluctuationsinpeakarea.Theyaretypicallycaused by malfunctioning check valves or air orcavitation in the pumphead. They are usually accompanied by fluctuations in retention times aswell. You can check if this is the problem by monitoring the backpressure. What fluctuations can betolerated depends onthe width of the peak. Assume thatthereis a flow difference of 10% between the two heads ofyour pump. If your peak width is about 20 pump strokes, the fluctuation in peak area caused by the pulsation of the pumpisunderl%. But it will be 10% if the peak width is aboutone pumpstroke.流速的随机变化也会导致峰面积波动。它们通常是由止回阀故障或泵头中的空气或气蚀引起 的。它们通常还伴随着保留时间的波动。您可以通过监控背压来检查这是否是问题所在。可 以容忍的波动取决于峰的宽度。假设您的泵的两个扬程之间的流量差为10%。假如您的峰 宽约为20个泵冲程，则由泵的脉动引起的峰面积波动小于1%。但是，假如峰值宽度约为 一个泵冲程，则它将为10%。Anotherpotential cause of flow-rate changes are leaks onthe high-pressure side of the system. They should be easy tofind.流速变化的另一个潜在缘由是系统高压侧泄漏。应当很简洁找到。Integrationerrors are more difficult to troubleshoot. Grosschanges in the way the software integrates the peaks can befound by simple visual examination, butmore subtle changesare difficultto spot. Significant integration errors often occurwith tailing peaks at a signal-to-noise ratio of 100 or less.Generally,thebase-linenoiselimitstheprecisionoftheintegration, but the problem becomesworse with fronting ortailing peaks.You can try to reprocess the data with differentsettings of the integration parameters and see if this reducestheproblem.积分错误更难以解决。通过简洁的目测即可发觉软件积分峰的方式的重大变化，但很难发觉 更微小的变化。信噪比为100或更低时，拖尾峰常常会产生明显的积分误差。通常，基线噪 声限制了积分的精度，但是随着峰前延或峰拖尾的消失，问题变得更加严峻。您可以尝试设 置不同的积分参数重新处理数据，然后查看是否可以解决问题。lncomplexsampleswhichcontainpeaksthatareonlymarginallyresolvedfromthepeaksofinterest,thes oft warecan have difficulties determining where the baseline is. Insuch a case, a manual integration or anautomatic integrationwith a forcedbaseline can improve the reproducibility of theintegration.在简单的样品中，其峰仅能从目标峰中略微辨别出来，因此该软件可能很难确定基线在哪里。 在这种状况下，使用强制基线进行手动积分或自动积分可以提高积分的可重复性。Thesample itself can be the source of the problem. Inreversed-phase chromatography it has been observed that some proteins donotelute completely during the in itial gradient. Anadditional quantity is eluted insubsequent blank gradients.This “ghosting” is specific to the protein and theelution protocol.Ifthis occurs,blank gradients need toberunbetweenanalyses.样本本身可能是问题的根源。在反相色谱中，已经观看到某些蛋白质在初始梯度过程中不会 完全洗脱。在随后的空白梯度中洗脱额外的量。这种“鬼峰”特定于蛋白质和洗脱方案。假 如发生这种状况，则需要在两次分析之间运行空白梯度。Also,proteinscanbe u irreversibly “ adsorbedonsomecolumns. The peak area obtained with abrandnew columnmay increase slowlywith repetitive injections. Presumably,someprotein binds to active sites on the packing. Once thesesites are saturated, a reproducible peak area is obtained. Itisnot necessary to use the proteinthat you need to analyze. Ithas beenreported that other proteins can be use as well tosaturatethe activesites.同样，蛋白质可以“不行逆地”吸附在某些色谱柱上。全新色谱柱获得的峰面积可能随着重 复进样而缓慢增加。据推想，某些蛋白质会与包装上的活性位点结合。这些地点一旦饱和， 获得了可重现的峰面积。不必使用您需要分析的蛋白质。据报道，也可以使用其他蛋白质来 饱和活性位点。The detector contributes to peak area fluctuations in anindirect way. How well the peak can be integrated depends on its signal-to-noise ratio. You can not expect are producibility of the peak area that isany better than theratio of the baseline noise to the signal at the peak maximum. Them obile phase is rarely acontribut or to peak area fluctuations. If there are any shifts in mobile phase composition, they will affect retentiontimes long before theyaffect peakareas. Ghost peaks or negative peaks that may begenerated by the mobile phase affect the integration in the same way as any other interference.They can of ten be avoided by dissolving the sample in the mobile phase.检测器间接地导致峰面积波动。峰的积分程度取决于其信噪比。您不能期望峰面积的重现性 比基线噪声与最大峰值处的信号的比率更好。流淌相很少是峰面积波动的缘由。假如流淌相 组成发生任何变化，那么它们将影响保留时间，而影响峰面积的时间要早得多。流淌相可能 产生的鬼峰或负峰同样影响积分。通常可以通过将样品溶解在流淌相中来避开这种状况。Q.：Whatreproducibility of the peak area can I reasonablyexpect?问：我可以合理预期峰面积的重现性吗？A.: Unlessyou are limited by the signal-to-noise ratio, therelative standard deviation of the peak area for repetitiveinjections should be definitely under l%,Values of 0.2 to0.5%areachievable.Thereproducibilitycanbefurtherincreased by using an internal standard and calculating theratio of the peak area of the analyte(s)to the peak area of theinternalstandard,butevenwiththismethodyou are notlikelyto beableto drivethe RSD muchunder 0.1%.答：除非您受到信噪比的限制，否则重复进样的峰面积的相对标准偏差确定应在1%以下。 可以达到0.2%到0.5%的值。通过使用内标并计算分析物的峰面积与内标的峰面积之比，可 以进一步提高重现性，但是即使使用这种方法，RSD也不太可能远远低于0.1%。