Ghost peaks in reversed phase gradient HPLC a review and (反相梯度HPLC鬼峰：综述和更新).docx

重庆东得医药科技有限公司 制剂分析部译Ghost peaks in reversed-phase gradient HPLC: a review and update反相梯度HPLC鬼峰：综述和更新Abstract摘要New examples and sources of ghost peaks in reversed-phase gradient HPLC are described and related to previous publications to give a broad perspective of ghost peak problems. 本文阐述了HPLC反相梯度鬼峰的新例子和原因，之前曾有相关刊物断言鬼峰问题将有一个广阔的前景。In one new example the ghost peak was found to be due to mixing problems caused by a period of non-delivery of the stronger “solvent B” in a stepped gradient system. 在一个新的例子中，鬼峰的出现是因为在分布梯度系统中一段时间未输入溶剂B而引起的。In a second example the ghost peak was due to plasticizer contamination of the organic solvent. 另一个案例中，鬼峰的出现是由于有机溶剂的塑化剂污染引起的。This paper includes tips and recommendations for the consistent running of ghost-peak-free reversed-phase gradient HPLC.本文讲述HPLC反相梯度持续运行过程中无鬼峰的提示和建议。Keywords: Gradient HPLC; Ghost peaks; System peaks; Chromatographic baselines; Water; Methanol; Acetonitrile; Solvents; TFA; Artifacts关键词：HPLC梯度，鬼峰，系统峰，色谱基线，水，甲醇，乙腈，溶剂，三氟乙酸，Contents目录1. Introduction 介绍2. Experimental 试验2.1. Instrumentation 仪器2.2. Materials 材料3. Results and discussion 结果和讨论3.1. Mobile phase contamination and the band compression mechanism 流动相污染及频带压缩原理3.1.1. Water 水3.1.2. Inorganic impurities in water 水中的无机杂质3.1.3. Organic solvents 有机溶剂3.1.4. Reagents and additives for mobile phases 流动相中的试剂和添加剂3.1.5. Phthalates, plastic additives and airborne organics as ghost peaks邻苯二甲酸盐，塑料添加剂和空气中的有机物成为鬼峰3.1.6. Contamination by mobile phase glassware 流动相玻璃容器的污染3.2. Other sources of ghost peaks 鬼峰的其他原因3.2.1. Pumping and mixing problems 泵和混合问题3.2.2. “Air peaks” by injection and mobile phase degassing problems 进样和流动相脱气引起的“气泡峰”3.2.3. Injection carryover 进样残留3.2.4. Systematic sample contamination 系统被样品污染3.2.5. Ghost peaks by mobile phase component interaction with the stationary phase 流动相成分与固定相相互作用3.3. Living with ghost peaks 鬼峰处理方式3.3.1. Electronic baseline subtraction 基线扣除4. Conclusions 结论Acknowledgements 致谢References 参考文献1. Introduction1.介绍In the highly regulated pharmaceutical industry controlis fundamental to all aspects of production and analysis.在高度规范化的制药行业，对于生产、检验的各个方面，“控制”是其根本。Reversed-phase gradient HPLC enables the analysis of a mixture of analytes possessing a broad range of polarity and retention characteristics in a single run, and for many applications this offers significant benefits over isocratic methodology.HPLC反相梯度能在单次运行里面分析具有广泛极性和保留特性的混合物，并且在许多应用里面都给出了超过等度方法的显著优势。However, gradient HPLC can sometimes be plagued with seemingly random and uncontrollable “ghost peak” problems, particularly if the equipment and materials available are not of very high quality, or if the analyst does not appreciate the sensitive mechanisms involved. 然而，HPLC梯度经常被看似随机和不可控制的“鬼峰”的问题所困扰，尤其是当设备和可用材料质量不是很高，或分析人员不能辨别其中的敏感机制。When ghost peaks suddenly appear in gradient HPLC chromatograms, the analyst may feel not in control of the analysis and may resort to less efficient isocratic methods that are ultimately more time-consuming and costly. 当鬼峰突然出现在梯度液相色谱中时，分析人员可能感到检验不受控制，并可采取效率较低的等度方法，那最终是更费时和昂贵的。However, if one understands the origins of ghost peaks and how to deal with them, then gradient HPLC can be run consistently and reliably. 但是，如果一个人理解鬼峰的起因以及如何处理它们，那么梯度HPLC可以被持续可靠地运行。Previously used terms in the literature include ghost peaks,artifact (and artefact) peaks 1, system peaks 2, pseudo peaks 3, vacancy peaks 4, eigen peaks 2,5, induced peaks 6, and spurious peaks 7 and this lack of common nomenclature makes researching the problem quite difficult 1. 在以前的文献中使用的术语包括鬼峰，人造（和人为）峰1，系统峰值2，伪峰3，空白峰4，特征峰2,5，引起峰6，和假峰7，这种缺乏共同命名使得研究问题相当困难1。“Ghost peak” now appears to be the most frequently used term 1,4,811 and will be used throughout this paper.现在看来“鬼峰”是最经常使用的术语1,4,8-11，并将在本文中使用，贯穿始终。There are many possible causes why ghost peaks may be observed in gradient HPLC, however almost always, only one common mechanism is responsible for their appearance. 有许多可能的原因导致为何鬼峰在梯度高效液相色谱中被观察到的，但是几乎总是只有一个共同的机制对它们的出现负责。This is that UV absorbing organic impurities in the mobile phase are focused into component bands on the column and subsequently eluted later in the gradient when the mobile phase possesses higher eluotropic strength. 这就是在流动相中UV吸收的有机杂质在色谱柱上被聚焦成组分带，并随后在梯度程序中被洗脱（当流动相具有较高的洗脱强度时）。The potential sources of these mobile phase impurities are wide-ranging. Other causes of ghost peaks include physical or mechanical aspects of mobile phase delivery, sample introduction and stationary phase effects.这些流动相杂质的潜在根源是广泛的。其他引起鬼峰的原因包括流动相输送的物理或机械方面，进样和固定相的影响。A chromatogram may contain ghost peaks from a variety of these sources and this can make the overallresolution of ghost peak problems quite difficult.色谱出现鬼峰的来源众多，这使得整个鬼峰问题的解决非常困难。In the following discussions, the impurity focusing mechanism is illustrated in more detail and some new and previously acknowledged causes are discussed along with tips for the identification and remediation of ghost peak problems.在接下来的讨论中，杂质聚焦机制将被更详细的说明。随着对鬼峰问题的识别和修复技巧，一些新的和以前认知的原因被进行了讨论。2. Experimental2.实验2.1. Instrumentation2.1. 仪器Three different HPLC systems were used in this work, specific conditions are described in the text. 在这项工作中使用了三种不同的HPLC系统，具体条件在文中所被描述。Figs. 2, 5, 7, 9 and 12 were obtained using a Waters 2695 chromatograph and Waters 2487 variable wavelength detector fromWaters Corporation (Milford, MA).图2，5，7，9和12是使用Waters Corporation (Milford, MA)的Waters2695色谱仪和Waters 2487可变波长检测器所获得。Figs. 3, 8, 11 and 13 were generated using an HP1100 system from Agilent Technologies Inc. (Wilmington,Delaware) comprising of a degasser (G1322A), quaternary pump (G1311A), autosampler (G1313A) and VWD detector (G1314A).图3，8，11和13是使用安捷伦科技公司（特拉华州威尔明顿）的HP1100系统产生。包括一个真空脱气机（G1322A），四元泵（G1311A），自动进样器（G1313A）和VWD检测器（G1314A）。Fig. 4 was obtained using a similar system, but with a binary pump (G1312A) rather than the quaternary pump.图4是使用一个类似的系统获得的，但采用的是一个二元泵（G1312A），而不是四元泵。Fig. 10 was obtained using aWaters 2795 and Waters 996 detector and accurate mass measurements were obtained online using a Micromass LC-TOF operating in +ESI mode,with the following settings: capillary voltage = 3000V, cone voltage = +25V, desolvation temp. = 250 C, source temp. = 120 C. Gas flows (N2); cone = 45 L/h, desolvation = 356 L/h.图10是使用Waters 2795 and Waters 996检测器获得。精确的质量测定是通过在线使用Micromass LC-TOF，在+ ESI模式操作时，使用以下设置获得。毛细管电压=3000V，锥电压=+25V，去溶剂化温度=250，源温度=120。气体流量（N 2）;锥体= 45升/小时，去溶剂化=356升/小时。UV spectra were obtained using a UV2-300 UVvis spectrometer from Unicam Ltd (Cambridge, UK), with a 1 cm quartz sample cell and an empty reference cell.紫外光谱是使用Unicam Ltd (Cambridge, UK)的UV2-300紫外可见光谱仪，以一个1厘米的石英样品池和空的参比池获得。Zorbax HPLC columns were supplied by Agilent Technologies Inc. (Wilmington, Delaware), Genesis columns were supplied by Jones Chromatography Ltd. (Hengoed, UK), Hichrom columns were supplied by Hichrom Ltd. (Reading, UK).Zorbax HPLC 色谱柱由安捷伦科技公司. (Wilmington, Delaware) 提供, Genesis 色谱柱由琼斯色谱有限公司(Hengoed, UK)提供, Hichrom色谱柱由 Hichrom Ltd. (Reading, UK)提供.2.2. Materials2.2. 材料Water for analysis was purified using a USF Elga Prima and Maxima unit supplied by Veolia Water Systems Ltd.(High Wycombe, Bucks, UK). 分析用水由Veolia Water Systems Ltd.(High Wycombe, Bucks, UK).提供的USF Elga Prima and Maxima unit纯化。Methanol, acetonitrile and tetrahydrofuran were obtained from various undisclosed UK suppliers. Formic acid (98/100%) was used as supplied by Fisher Scientific (Loughborough, UK). 甲醇，乙腈和四氢呋喃分别从各个未公开英国供应商获得的。甲酸（98/100）由Fisher Scientific公司提供。Trifluoroacetic acid (99% grade and spectrophotometric grade) and dioctyl phthalate 99% (as bis-(2-ethylhexyl) phthalate) were used as supplied by SigmaAldrich (Gillingham, Dorset, UK).三氟乙酸（99等级和分光级）和苯二甲酸二辛酯99（如双 - （2-乙基己基）邻苯二甲酸酯），由Sigma-Aldrich公司提供。3. Results and discussion3.1. Mobile phase contamination and the band compression mechanism3.结果与讨论3.1流动相污染和频带压缩原理In all chromatography longitudinal diffusion serves to increase the bandwidth of the separating components. 在色谱洗脱过程中，由于样品的延展，这些组份会形成一定的带宽。In isocratic HPLC, one tries to elute the components before the longitudinal diffusion becomes unmanageable and the peaks are too broad.在等度HPLC中，一种组份在纵向扩散过程中，峰是较宽的。In gradient elution chromatography, there is an active focusing mechanism (band/peak compression) that dynamically compresses the component bands.在梯度HPLC中，就有一种聚集机制（带/峰压缩），即单位频带会不断的进行压缩。The peak width observed in a gradient HPLC run is the product of synchronous diffusion and focusing processes. 在梯度HPLC运行中，所得到组份的峰宽是不断扩散、聚集得到的结果。In a linear solvent gradient, the peak width should be approximately constant throughout the run 12.在线性梯度程序中，各组分的峰宽基本是恒定不变的12。The focusing mechanism can be rationalised as follows：imagine a component band broadly diffused on the head of an HPLC column. 对这种聚集机理的合理解释是，假设有单位宽度的物质（即频带）从HPLC色谱柱柱头处开始进行洗脱。When a solvent gradient is applied the concentration of organic solvent is always higher at the back of the band than at the front of the band. 当进行梯度洗脱时，频带后面的有机相浓度总是大于频带前端的。Hence, the component molecules at the front of the band are more strongly retained by the stationary phase than those at the back. 因此，该频带前端的物质在固定相中保留的能力比在后端的物质强。As such, the component molecules at the back of the band are always more mobile, any that lag behind are immediately picked up by the stronger eluent and moved forward more quickly to catch up with the rest. 这样，在该频带后端的物质分子是总是移动得更快，任何在后面的组份，均由于洗脱液更强，所以可以更快速的追上其余部分。Provided that the column is long enough and the gradient is steep enough, peak focusing (or compression) progresses up to the point whereby the balance of diffusive and focusing processes determines the final peak width, Fig. 1. 当然，前提条件是该色谱柱足够长且梯度足够剧烈，峰聚集（或称为压缩）最终会达到平衡，此时得到最终的峰宽，如图1所示。Fig. 1. Schematic of a component band being compressed through a solvent gradient of increasing eluotropic strength. The % methanol figures are arbitrary values to illustrate that a higher methanol concentration always prevails at the back of the component band irrespective of its position in the column.图1：图示为单位频带的物质的梯度中的洗脱情况。其中甲醇（）的数值是随意设置的，只是为了表明在色谱柱中的任何位置，频带后端洗脱液浓度比前端高。Longitudinal diffusion is minimised by using smaller diameter spherical particles, e.g. 3m rather than 5m silica. 这种纵向扩散在越小的球形颗粒中效果越好，例如，3m的硅胶颗粒，最多不超过5m。The band compression mechanism is maximised by increasing the gradient steepness.同时梯度变化越剧烈，频带压缩的效果越好。The band compression mechanism may similarly focus organic impurities present in the mobile phase. 这种频带压缩原理就类似于流动相中有机杂质的聚集情况。Impurities that show some retention at low-eluent strength may be focused into peaks as the gradient progresses. 这表明有些在低强度洗脱液中保留较强的杂质可随梯度洗脱逐渐聚集。This same “trace enrichment” mechanism is exploited in environmental analysis, where pesticides and organic residues are loaded onto a column from a large volume of aqueous sample and then eluted with a focusing solvent gradient 13.此“微量富集”机制常用于环境分析，将含有杀虫剂和有机残留的大量水样过柱，进行洗脱富集 13。After sufficient focusing, mobile phase ghost peaks appear to be identical to injected analyte peaks, however, where focusing is not complete they may be broader or possess an atypical shape.经过充分富集，在进样分析中，会得到相似的流动相鬼峰，当然，鬼峰产生的原因并不完全是这种聚集的原因，可能还有其他不明因素。Fig. 2 shows how by a focusing mechanism, impurities at levels around 1ng/mL in the mobile phase may be observed in a blank gradient run. 图2显示了如何通过上述的聚集机理，使浓度在1ng/ml水平杂质，可以在空白基线中显示出来。Fig. 2. Chromatogram of eluent spiked with dioctyl phthalate (DOP), Column: Zorbax XDB-C8, 150 mm 4.6 mm, 5m, temp. 40, 1.5 mL/min， = 265 nm. Eluent A: water + 1 ng/mL DOP (as bis-2-ethylhexyl phthalate); eluent B: acetonitrile. Timetable (mins, %B): (0, 20), (15, 95), (17,95), (17.1, 20), (22, 20).图2：流动相中掺入邻苯二甲酸二辛酯（DOP）；柱：XDB Zorbax-C8，150mm4.6mm，5m；温度40；1.5ml/minn；=265nm；洗脱液A：水+1ng /ml DOP；洗脱剂B：乙腈；时间表（分钟，B）：（0，20），（15，95），（17,95），（17.1，20），（22，20）。Although this dioctyl phthalate ghost peak is small, it may still interfere with the quantification of low-level organic impurities in a pharmaceutical analysis.虽然此邻苯二甲酸二辛酯鬼峰很小，它仍可能干扰药品中微量杂质定量检测。The ideal gradient HPLC mobile phase arriving at the top of the column should contain only the intended solvents and reagents and absolutely nothing else.理想的HPLC梯度流动相，在到达色谱柱前端时，应只含有溶剂和待测物质，而不应该有其他物质。Gradient HPLC is not only the analysis of the sample, but also an analysis of the impurities of the mobile phase and solvent pathway of the instrument. 在梯度HPLC中得到的峰，不仅仅是样品峰，还包含有流动相以及仪器管路中的杂质峰。To distinguish real analyte peaks from these system (or ghost) peaks, the examination of blank injections is essential.所以为了区分样品峰与系统峰（鬼峰），必须进行空白考察。3.1.1. Water3.1.1水Water used for gradient HPLC must be as pure and fresh as possible. It should ideally be free from trace organics, inorganics and particulates. 用于梯度HPLC的水必须尽可能的纯净、新鲜。最好不含微量有机物、无机物及其它颗粒物质。Fortunately, for the busy pharmaceutical analyst commercially available equipment can supply the needs of the laboratory such that the water should not be a concern, however, to get good results these purifier systems must be set up and maintained to high standards.好在，目前已能买到满足实验室要求的纯水机，但是，这些设备需要良好的维护。Milli-Q and Elga Maxima units are examples of systems that can provide such quality. 美国Millipore公司的Milli-Q系列纯水机、威立雅公司的Maxima纯水制备装置均能提供符合上述质量要求的水。Essential components of these systems are first a heavy, activated carbon and 5m pre-filter (to remove particulates and lower the chlorine content to sub-g/mL levels), a reverse osmosis unit (to remove the majority of ionic species) and ultraviolet photo-oxidation to kill bacteria and oxidize organic species 11,14. 组成这些系统的第一部分为活性炭和孔径为5m的过滤器（以除去颗粒物质并降低氯化物含量至g/mL的水平），以及一个反渗透单元（以除去大部分离子物质）和紫外线装置（杀死细菌和氧化有机物质）11,14。Ion-exchange and carbon adsorption media are used to further lower the inorganic and organic content and an ultra-microfiltration unit (0.05m on Elga Maxima) is employed to ultimately remove bacteria and fine particulates. 随后的离子交换装置和碳吸附介质可进一步降低无机和有机物质的含量，最后采用超精密过滤单元（Elga Maxima为0.05m）来最终除去细菌和极细微的颗粒。The system should be disinfected and the consumable parts replaced at regular intervals. Intermittent recycling should also be operating to regularly re-purify water that stands in the system.该系统应定期进行消毒和耗材的更换，并且每隔一段时间需对净水系统进行循环清洗。There are many other possible approaches to producing pure water, but few are as practical or effective as the commercial systems described above. 当然还有许多其他方法可用来生产纯净水，但都没有上述系统实用或有效。Several authors have discounted distillation for reasons of cost and efficiency 1418 and many low-molecular weight organic compounds carry over in the distillation. 出于成本和制备效率的考虑，有人采用折中的蒸馏法，可通过蒸馏除去小分子有机化合物1418。Homemade attempts at purification by reversed-phase impurity adsorption often fail because the purification media used is more loaded with impurities than the water itself 19,20. 采用反相吸附法自制纯化水常常不能获得成功，是因为所采用的净化设施中含有更多的杂质。In addition, adsorption systems may require too much maintenance to run long-term and the breakthrough of impurities always ultimately occurs 18. 除此之外，采用吸附装置的净化系统需要长期进行维护，不然最终杂质也会超标18。The most practical proposal to date has been described by Ringo 21 who recommends the use of disposable polystyrene divinylbenzene extraction disks in standard solvent filtration apparatus. 最实用的方法是由Ringo21提出来的，他建议在标准溶剂过滤装置中使用一次性的聚苯乙烯二乙烯基苯过滤盘。These disks absorb hydrophobic UV active organic compounds from water and aqueous buffer solutions.这些过滤盘可以从水或含水缓冲盐中吸附具有紫外吸收的物质。The size and format of these disposable disks means that they can be easily replaced, or regenerated with clean organic solvent.同时根据实际情况，可更换这种一次性过滤盘的大小和尺寸，并且还可以采用有机溶剂清洗后进行再生利用。McCown et al. 18 have reported that continuous degassing with helium (48 h) or prolonged boiling (3 h) significantly reduces the amount of low-boiling impurities, however these would be impractical procedures for many users and neither approach removes high-boiling impurities.麦科恩等人报道18，用氦气脱气（48h）或长时间沸腾（3h）可显著减少低沸点杂质，但是这些方法对许多人并不适用，而且还不能除去高沸点的杂质。Some in-line HPLC mobile phase purification systems have been shown to work effectively, but these generally necessitate the use of a binary pump system and in-line switching valves to enable back-flushing of the filter media 22,23.一些HPLC仪器的在线过滤器可起到很好的作用，但这通常需要使用二元泵或比例阀，以便对过滤装置进行反冲洗。Fig. 3. Comparison of a clean and contaminated sequential isocratic step chromatogram. 图3.干净的与被污染的梯度洗脱色谱图比较。Column: Genesis C18, 150mm 4.6 mm, 4 _m, temp.25 C, 1.5 mL/min, = 235 nm. Eluent A: 750 methanol, 250 water; eluentB: 900 methanol, 100 water.色谱柱：Genesis C18, 150mm 4.6 mm, 4um，柱温25，流速1.5 mL/min,波长235nm。流动相A：甲醇：水（75：25），流动相B：甲醇：水（90：10）Timetable (min, %B): (0, 0), (45, 0), (46,100), (60, 100), (60.1, 0), (70, 0).梯度：时间（min） 0 45 46 60 60.1 70 B% 0 0 100 100 0 0Fig. 3 demonstrates the sensitivity of gradient HPLC systems to water quality. 图3展示了梯度HPLC系统对水质的敏感性.The lower trace represents a pharmaceutical related substances method using a “sequential isocratic step gradient” of 75% methanol, stepped up to 90% methanol after 45 min. 下方迹线代表使用75的甲醇， 45min后甲醇加至90%的药物有关物质 “梯度洗脱”方法。The upper trace is the same method exhibiting a severe gradient ghost peak that renders the analysis useless.上方迹线是用相同方法出现严重的呈现分析无用的梯度鬼峰。The contaminants begin to break through towards the end of the first isocratic period, and are finally washed out as the eluent strength increases.污染物在第一个等度期结束时开始出现，在洗脱强度增大时被洗脱。The reason for this gross system contamination was not fully confirmed, but was strongly suspected to be due to a neglected and bacterially infected water purifier system. 系统污染的总体原因没有得到充分的证实，但是很有可能是因为忽略了净水器系统被细菌感染。The very broad retention range of the impurities is commensurate with the var