ACCQ Prep制备色谱中聚焦梯度检测方案(制备液相色谱)

EverywhereyoulookTN54 Chromatography Technical Note TN54P.O. Box 82531， Lincoln， Nebraska， 68501 USAToll-free：(800) 228-4373·Phone： (402) 464-0231·Fax：(402) 465-3091Teledyne ISCO is continually improving its products and reserves the right to change productspecifications， replacement parts，schematics， and instructions without notice.Mar 2021 Global Calibration Function ofAnalytical to Different ColumnChemistries@ on the ACCQPrep TELEDYNE ISCO Chromatography Technical Note Abstract Chemists often evaluate different column chemistries for compound purification during analytical scoutingruns. For example， it is common for chiral purification，for several column types to be screened on an analyticalHPLC. Despite very different types of columns， withwidely varying chemistry， the ACCQPrep can calculatefocused gradients from them with only a singlecalibration on one column type. Although the ACCQPrepuses a global calibration for all columns installed on thesystem， that calibration is not limited to a single columnchemistry. The global calibration can also be used fordifferent column chemistries， so long as the analyticalcolumns have the same dimensions and use the samegradient method table and flow rate， allowing fast andeasy column and method screening. Background Many labs， such as those performing chiral purifications，do method development using many types ofcolumnsand solvents. The method development starts witha scouting run which allows an initial evaluation ofretention and resolution.The scouting runs can thenbe used to create focused gradients for the ACCQPreppreparative HPLC system. The ACCQPrep preparativeHPLC has a built-in calibration for the scouting methodcalculated by PeakTrak software when columns areinstalled. This built-in calibration works for all columns，regardless of manufacturer. The external HPLCcalibration in the ACCQPrep can likewise be used for avariety of column chemistries provided the analyticalcolumns are the same dimensions， and the samegradient table is used for all columns. Experimental and Results C18 runs with a C8 calibration Universal Test Mix (PN 60-5234-835) was run on aRediSep Prep C8 column (20x150 mm， 5u， 200A， PN69-2203-858) to get a retention of 6 minutes for the firsteluting peak using a methanol/water solvent system， asdescribed in Technical Note 52 (TN52)， Calibration ofAnalytical LC systems available on the Teledyne ISCOweb site. 一o Time (Minutes) Figure 1-Calibration of Teledyne ISCO C8 columns on theACCQPrep(top)，and UHPLC (bottom). A matching C8 UHPLC column (2x50 mm， PN 69-2203-853) was run in a water/methanol gradient (5 to 100%B over 5 minutes， with an isocratic hold for 2.5 min at100% methanol). The peak eluting at 3.757 minutes，corresponding to the peak eluting at 6 minutes on theACCQPrep， was used for the calibration into PeakTrakin the ACCQPrep. Preparative runs The C8 analytical column was replaced with ananalytical RediSep Prep UHPLC C18 column (2x50mm， PN 69-2203-854) and test mix was run in a water/methanol gradient using the same gradient as used forthe C8 column. As expected， the compounds eluted laterin the gradient due to the increased retention of the C18column， with 100 A pores. 3500 - o 广oc 二oc 0 2 4 6 8 10 12 14 16 Time (Minutes) Figure 2-Analytical scouting runs and focused gradientson a C18 column， using the C8 column calibration. The firstpreparative run was focused on the compound eluting at4.324 minutes in the analytical run， while the peak at 4.953minutes was used to calculate the second preparative gradient The data from the C18 analytical run was used tocalculate the preparative gradients in Figure two whileusing the calibration from the C8 columns in Figure 1.The preparative purifications utilized a RediSep PrepC18 column (20x150 mm， 5u， 100A， PN 69-2203-810) Inboth cases， the compounds eluted close to the expected6-minute elution time. The test mix was also run on the C8 preparative HPLCcolumn with the C8 calibration； the analytical data inFigure 1 was used to calculate the gradient for the secondeluting peak at 4.451 minutes. oc Figure 3-Preparative run on a RediSep Prep C8 columnusing the second compound eluting from the analytical runin Figure 1. The chromatogram appears almost the same as the C18column， the only difference is the solvent compositionat the start and end of the focused gradient due to thedifferences in retention between the two columns. Silica runs with a C18 calibration A 4.6x150 mm RediSep Prep C18 column was calibratedon an Agilent analytical HPLC sys1.tem as described inTN52 using a gradient from 5 to 100% methanol over 6minutes， followed by 100% methanol for 6 minutes at 1.0mL/min. The solvent was changed to hexane/ethyl acetate11mnranlacad sxand the column replaced with a 4.6x150 mm RediSepPrep silica column. The same gradient method was usedfor the new solvent and column， 5 to 100% ethyl acetateover 6 minutes. A sample of methyl paraben was injected，and the retention time used for the focused gradientshown in Figure 4 on a 20x150 mm RediSep Prep silicacolumn run in hexane/ethyl acetate. The sample elutednear the middle of the calculated gradient. Figure 4-Scouting run and calculated focused gradient ofa silica column using a calibration for a C18 column. Conclusion The calibration used for one column chemistry can beused for another column chemistry in the PeakTrakFocused Gradient Generator. This allows greaterflexibility to choosing columns for the analytical andpreparative systems and ensuring proper scalability.Follow the simple rule where the analytical columnsused with the external HPLC need to be the same sizewith respect to length and inner diameters and runwith the same gradient table and flow rate. This allowsa user to use analytical scouting runs with differentcolumns and solvent systems to determine the bestpurification method with higher loading capacity. ( Teledyne ISCO ) 摘要在分析方法考察过程中，化学家经常评估纯化过程化合物在不同色谱柱下的化学特性，例如，对于手性化合物的纯化，通常在高效液相色谱分析中筛选几种类型的色谱柱，尽管不同类型的色谱柱，有着很大的化学差异性，尽管ACCQ Prep制备色谱系统上可以安装、使用各种类型色谱柱，ACCQ Prep可以通过只需对一种类型的色谱柱单次校准计算出聚焦梯度，该校准不局限于单个色谱柱，通用校准也可以用于不同化学特性的色谱柱，只要分析型色谱柱有相同的尺寸和使用相同的梯度和流速，就允许快速和简便的色谱方法筛选。背景许多实验室，例如那些进行手性物质纯化的实验室，使用多种类型的色谱柱和溶剂进行方法开发，一般从对保留时间和分离度的初步评估开始，然后，侦测运行可以用来创建ACCQ Prep聚焦梯度的高效液相色谱制备方法，色谱柱安装后，ACCQ Prep制备型HPLC的PeakTrak®软件具有内置校准、计算侦测方法，这种内置校准的工作适用于所有色谱柱，无论色谱柱的制造商。ACCQPrep外部HPLC校准同样可以用于各种类型色谱柱，只要分析柱的尺寸相同，并且使用相同的梯度。实验和结果C18色谱柱运行与C8色谱柱校准通用测试混合物(PN 60-5234-835)在RediSep® C8制备色谱柱(20x150mm,5µ,200Å,PN 69-2203-858)上运行，使用甲醇/水溶剂系统，得到第一个保留时间在6min的洗脱峰，如在Teledyne ISCO 网站Technical Note 52 (TN52)所述的《校准分析型HPLC系统》。相匹配的C8 UHPLC色谱柱(2x50 mm, PN 69-2203- 853)在水/甲醇体系中运行(5min 5 - 100% B，在100%甲醇下等度保持2.5min)，峰洗脱时间为3.757min，对应于ACCQPrep上的峰洗脱时间为6min。制备色谱运行C8分析柱更换为RediSep Prep UHPLC C18分析柱(2x50 mm, PN 69-2203-854)，测试混合物在水/甲醇梯度中运行，梯度与C8柱相同。正如预期的那样，由于C18色谱柱填料100Å孔径使得保留增加，化合物在梯度中较晚被洗脱。使用C18分析运行的数据计算图2中的制备型梯度，同时使用图1中的C8色谱柱的校准。制备纯化是采用RediSep Prep C18柱(20x150 mm, 5µ，100Å， PN 69-2203-810)。两种情况下，化合物洗脱时间都接近预期的6分钟洗脱时间。同时在C8制备型高效液相色谱柱上运行测试混合物，并进行C8校准；利用图1中的分析数据计算第2洗脱峰在4.451min时的梯度。色谱图看起来与C18柱几乎相同，唯一的区别是由于两色谱柱之间的保留差异聚焦梯度开始和结束的溶剂组成不同。硅胶色谱柱运行与C18校准4.6 × 150 mmRediSep Prep C18柱在AgilentHPLC分析系统上进行校正，如TN52中所述，梯度6min内从5到100%甲醇，然后以1.0mL/min的流速100%甲醇保持6min。溶剂改为正己烷/乙酸乙酯，色谱柱改为4.6 × 150 mm RediSep Prep硅胶柱。同样的梯度法用于新溶剂体系和色谱柱，梯度6min内5至100%乙酸乙酯，将methyl paraben样品注入，在20x150mm的RediSep Prep硅胶柱上，在正己烷/乙酸乙酯中进行聚焦梯度的保留时间如图4所示，样品在计算梯度的中间附近被洗脱。结论在PeakTrak聚焦梯度生成中，对于某类化学特性的色谱柱校准可用于另一类化学特性的色谱柱，这允许在选择分析、制备系统色谱柱的更多灵活性，并确保适当的可扩展性。遵循一个简单的规则，在使用外部HPLC分析柱的长度和内径尺寸相同情况下，然后运行具有相同的梯度和流量。这允许用户使用分析型HPLC侦测运行不同的色谱柱和溶剂系统，以确定具有较高样品承载能力的最佳净化方法。ACCQ Prep HP150 高压制备型HPLC色谱系统HP150直观、易于使用、简单纯化设计理念，用户界面友好，消除了普通高效液相色谱系统中不必要的和复杂的参数设置。提高高效液相制备色谱性能和准确性，提高纯化样品回收率。内置馏分收集器和集成触摸屏，紧凑设计节省空间，HP150系统技术特点：流速1-150 mL/min开发分析方法和制备方法操作压力可达6000 psiUV或UV/VIS基础上选择ELSD和MS检测器一键生成聚焦优化梯度纯化时间最小化，样本回收率最大化