One of the authors (DK) acknowledges the receipt of an Australian Postgraduate Award.

注意:请参考所有材料和试剂材料安全数据表（MSDS）使用前（ 即 MSDS甲醇）。处理溶剂和高效液相色谱法（HPLC）洗脱时，确保使用的所有适当的安全措施。确保适当使用高效液相色谱仪，分析天平和检测仪器仪表工程控制，保证使用个人防护用品（防护眼镜，手套，实验室外套，全长裤，封闭趾鞋）。 注意:此协议包含如何耦合用UV-Vis检测器HPLC系统上使用CF列的指令。该协议已被写入假设读者已经在色谱基础知识和经验。 1. HPLC仪器的安装注意:本节可以改变，以适应分析人员的需求， 即，选择的溶剂中，检测器波长和流动速率适合于所关心的样本。 <ol><li>制备具有100％的超纯水为线，一种用于线B的高效液相色谱仪（ 例如 Milli-Q水）和100％甲醇作为流动相和吹扫泵按照制造商的要求。 </li><li>在UV-Vis检测设定为254纳米。 </li><li>选择的设置任一个预喷射流分割模式，或一个双泵流量设置。对于分流模式进行第2步，为双泵模式继续执行步骤3。 </li></ol> 2.分流系统设置<ol><li>断开从自动取样器的喷射器阀的泵线。 </li><li>附加一个T型件到泵线。 </li><li>附上15厘米的一块0.13毫米ID管到T型件的每一个端口。 </li><li>从T型件连接一管喷射器自动取样的阀。 </li><li>泵设置至1.0ml分钟-1。 </li><li>流的分割比连接泵线到CF柱的入口，调谐到40％之前:6<html> 0％（中线:外围线）在步骤如下2.7。 </li><li> CF的比例入口分流系统的调整<ol><li>用分析天平测量两个空收集容器的质量和标签一个收集容器的中心和另一外围设备（一个用于从自动取样器行居中端口，一个用于从T型件到外围端口的线） 。 </li><li>为1.0分钟，收集排出的流动相从所述喷射器（在将被连接到所述列中的点）进入收​​集容器中，在2.7.1测定其质量来的行。 </li><li>重新称量在分析级的收集容器中，并确定所收集的流动相的质量。 </li><li>重复步骤2.7.2至2.7.3用于退出从T型件就是要连接到外围端口行洗脱剂。 </li><li>确定流量的比例（毫升分钟-1）从根据下列等式流的每个行: 1"SRC ="/文件/ ftp_upload / 53471 / 53471eq1.jpg"/&gt; </li><li>调整流量比例为40％:60％（±2％）（线从注射器到中央端口:T-片外围端口线）。如果从喷射器到中央端口的流量百分比的线是40％以上，提高通过减小管的内直径或增加其长度的压降。如果从喷射器到中央端口的流量百分比的线是在40％以下，提高了管子的内径或减小管的长度。 </li><li>一旦流动比率调整转向泵的流量了。 </li><li>从喷油器线路连接到柱入口的中心港口，从T-片柱入口的外设端口的线。 </li><li>缓慢上升流速至1.0ml分钟-1在100％B线。 </li><li>通过允许平衡所述柱（4.6毫米内径×100毫米长）100％甲醇（B线）流动相以1.0ml分钟通过该柱流-1 10分钟。这个时间是根据用户可以使用其他列的尺寸缩放。 </li><li>对于CF出口的调谐转到步骤4"CF出口流量的调整"。 </li></ol></li></ol> 3.双泵系统设置<ol><li> HPLC系统泵连接到喷射器，然后从喷射器连接线到该列的中央进气口。 </li><li>直接连接的附加泵到柱的入口外围端口。注意，该第二泵由绕过喷射器。 </li><li>斜坡连接到中央口系统泵的流速0.4毫升分钟-1（代表为1.0ml分钟-1的总流速的40％），在100％甲醇（B线）。 </li><li>同时如步骤3.3，坡道的周边泵的流速0.6毫升分钟-1（代表的总流量的60％1.0毫升分钟-1），在100％甲醇（B线）。 </li><li>通过允许100％甲醇（B线）的流动相以1.0ml分钟通过该柱流-1 10分钟平衡的柱（4.6毫米内径×100毫米长）。这个时间是根据用户可以使用其他列的尺寸缩放。 </li><li>对于CF出口的调谐转到步骤4"CF出口流量的调整"。 </li></ol> 4.调整CF出口气流<ol><li>中央出口端口连接到使用15厘米的一块0.13毫米ID管的UV-Vis检测。 </li><li> 15厘米的一块0.13毫米ID管连接到CF柱的外周出口。 </li><li>权衡的分析天平上两个空收集容器的质量和标签一个容器的中心和其他外围设备。 </li><li>为1.0分钟，收集从UV-Vis检测器（中央流动）排出的流动相进入收集容器标签的中心，在4.2测定其质量。 </li><li>重新称量含有关于分析级所收集的洗脱剂在收集容器中，并确定所收集的流动相的质量。 </li><li>重复步骤退出从外围出口线路洗脱液4.4至4.5。 </li><li>确定流从根据下列等式流中的每一行的百分比: <img alt="公式（2）" src="/files/ftp_upload/53471/53471eq2.jpg" /></li><li>调整的流量比至21％:79％（±2％）（从紫外可见中心出口流量:从管线外围出口流），如果从紫外可见中央流百分比为21％以上，提高压降通过减少附着到UV-Vis检测的出口管道的内部直径或增加其长度。如果从紫外可见的中心流动百分比低于21％，增加内径附着在UV-Vis检测出口处的管道，或减小管子的长度。每次管的长度被改变，重复步骤4.3至4.7。 注:"虚拟"2.1 mm内径模式CF柱为分析做好准备。 </li></ol>

<ol><li>Camenzuli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+parallel+segmented+outlet+flow+columns+for+enhanced+mass+spectral+sensitivity+at+high+chromatographic+flow+rates%5BTitle%5D)+AND+%22Rapid+Commun.+Mass+Sp%22%5BJournal%5D)">The use of parallel segmented outlet flow columns for enhanced mass spectral sensitivity at high chromatographic flow rates.</a> Rapid Commun. Mass Sp. 26 (8), 943-949 (2012).</li>
<li>Camenzuli, M., Ritchie, H. J., Ladine, J. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+separation+performance+using+a+new+column+technology%3A+Parallel+segmented+outlet+flow%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Enhanced separation performance using a new column technology: Parallel segmented outlet flow.</a> J. Chromatogr. A. 1232 (0), 47-51 (2012).</li>
<li>Camenzuli, M., Ritchie, H. J., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gradient+elution+chromatography+with+segmented+parallel+flow+column+technology%3A+A+study+on+4.6mm+analytical+scale+columns%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Gradient elution chromatography with segmented parallel flow column technology: A study on 4.6mm analytical scale columns.</a> J. Chromatogr. A. 1270 (0), 204-211 (2012).</li>
<li>Camenzuli, M., Ritchie, H. J., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+HPLC+separation+performance+using+parallel+segmented+flow+chromatography%5BTitle%5D)+AND+%22Microchem.+J%22%5BJournal%5D)">Improving HPLC separation performance using parallel segmented flow chromatography.</a> Microchem. J. 111 (0), 3-7 (2013).</li>
<li>Camenzuli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parallel+segmented+outlet+flow+high+performance+liquid+chromatography+with+multiplexed+detection%5BTitle%5D)+AND+%22Anal.+Chim.+Acta%22%5BJournal%5D)">Parallel segmented outlet flow high performance liquid chromatography with multiplexed detection.</a> Anal. Chim. Acta. 803 (0), 154-159 (2013).</li>
<li>Kocic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+through-put+and+highly+sensitive+liquid+chromatography-tandem+mass+spectrometry+separations+of+essential+amino+acids+using+active+flow+technology+chromatography+columns%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">High through-put and highly sensitive liquid chromatography-tandem mass spectrometry separations of essential amino acids using active flow technology chromatography columns.</a> J. Chromatogr. A. 1305 (0), 102-108 (2013).</li>
<li>Shalliker, R. A., Ritchie, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Segmented+flow+and+curtain+flow+chromatography%3A+Overcoming+the+wall+effect+and+heterogeneous+bed+structures%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Segmented flow and curtain flow chromatography: Overcoming the wall effect and heterogeneous bed structures.</a> J. Chromatogr. A. 1335 (0), 122-135 (2014).</li>
<li>Shellie, R., Haddad, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+two-dimensional+liquid+chromatography%5BTitle%5D)+AND+%22Anal.+Bioanal.+Chem%22%5BJournal%5D)">Comprehensive two-dimensional liquid chromatography.</a> Anal. Bioanal. Chem. 386 (3), 405-415 (2006).</li>
<li>Abia, J. A., Mriziq, K. S., Guiochon, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radial+heterogeneity+of+some+analytical+columns+used+in+high-performance+liquid+chromatography%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Radial heterogeneity of some analytical columns used in high-performance liquid chromatography.</a> J. Chromatogr. A. 1216 (15), 3185-3191 (2009).</li>
<li>Knox, J. H., Laird, G. R., Raven, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+radial+and+axial+dispersion+in+liquid+chromatography+in+relation+to+the+%26%2334%3Binfinite+diameter+effect%26%2334%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Interaction of radial and axial dispersion in liquid chromatography in relation to the &#34;infinite diameter effect&#34.</a> J. Chromatogr. A. 122 (0), 129-145 (1976).</li>
<li>Miyabe, K., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estimation+of+the+column+radial+heterogeneity+from+an+analysis+of+the+characteristics+of+tailing+peaks+in+linear+chromatography%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Estimation of the column radial heterogeneity from an analysis of the characteristics of tailing peaks in linear chromatography.</a> J. Chromatogr. A. 830 (1), 29-39 (1999).</li>
<li>Shalliker, R. A., Scott Broyles, B., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Axial+and+radial+diffusion+coefficients+in+a+liquid+chromatography+column+and+bed+heterogeneity%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Axial and radial diffusion coefficients in a liquid chromatography column and bed heterogeneity.</a> J. Chromatogr. A. 994 (1-2), 1-12 (2003).</li>
<li>Gritti, F., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+the+thermal+heterogeneity+of+the+column+on+chromatographic+results%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Effects of the thermal heterogeneity of the column on chromatographic results.</a> J. Chromatogr. A. 1131 (1-2), 151-165 (2006).</li>
<li>Shalliker, R. A., Wong, V., Broyles, B. S., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+bed+compression+in+an+axial+compression+liquid+chromatography+column%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Visualization of bed compression in an axial compression liquid chromatography column.</a> J. Chromatogr. A. 977 (2), 213-223 (2002).</li>
<li>Tallarek, U., Albert, K., Bayer, E., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+transverse+and+axial+apparent+dispersion+coefficients+in+packed+beds%5BTitle%5D)+AND+%22AICHE+J%22%5BJournal%5D)">Measurement of transverse and axial apparent dispersion coefficients in packed beds.</a> AICHE J. 42 (11), 3041-3054 (1996).</li>
<li>Camenzuli, M., Ritchie, H. J., Ladine, J. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Active+flow+management+in+preparative+chromatographic+separations%3A+A+preliminary+investigation+into+enhanced+separation+using+a+curtain+flow+inlet+fitting+and+segmented+flow+outlet%5BTitle%5D)+AND+%22J.+Sep.+Sci%22%5BJournal%5D)">Active flow management in preparative chromatographic separations: A preliminary investigation into enhanced separation using a curtain flow inlet fitting and segmented flow outlet.</a> J. Sep. Sci. 35 (3), 410-415 (2012).</li>
<li>Shalliker, R. A., Broyles, B. S., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physical+evidence+of+two+wall+effects+in+liquid+chromatography%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Physical evidence of two wall effects in liquid chromatography.</a> J. Chromatogr. A. 888 (1-2), 1-12 (2000).</li>
<li>Shalliker, R. A., Camenzuli, M., Pereira, L., Ritchie, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parallel+segmented+flow+chromatography+columns%3A+Conventional+analytical+scale+column+formats+presenting+as+a+%27virtual%27+narrow+bore+column%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Parallel segmented flow chromatography columns: Conventional analytical scale column formats presenting as a 'virtual' narrow bore column.</a> J. Chromatogr. A. 1262 (0), 64-69 (2012).</li>
<li>Camenzuli, M., Ritchie, H. J., Ladine, J. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+design+of+a+new+concept+chromatography+column%5BTitle%5D)+AND+%22Analyst%22%5BJournal%5D)">The design of a new concept chromatography column.</a> Analyst. 136 (24), 5127-5130 (2011).</li>
<li>Foley, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precision+and+Reliability%3A+an+Intercontinental+Study+of+Curtain+Flow+Chromatography%5BTitle%5D)+AND+%22Thermo+Scientific%22%5BJournal%5D)">Precision and Reliability: an Intercontinental Study of Curtain Flow Chromatography.</a> Thermo Scientific. , (2013).</li>
<li>Pravadali-Cekic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multidimensional+Approaches+for+the+Analysis+of+Complex+Samples+using+HPLC%5BTitle%5D)+AND+%22University+of+Western+Sydney%22%5BJournal%5D)">Multidimensional Approaches for the Analysis of Complex Samples using HPLC.</a> University of Western Sydney. , PhD thesis (2014).</li>
<li>Soliven, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+the+performance+of+narrow-bore+HPLC+columns+using+active+flow+technology%5BTitle%5D)+AND+%22Microchem.+J%22%5BJournal%5D)">Improving the performance of narrow-bore HPLC columns using active flow technology.</a> Microchem. J. 116 (0), 230-234 (2014).</li>
<li>Foley, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Curtain+flow+chromatography+%28%27the+infinite+diameter+column%27%29+with+automated+injection+and+high+sample+through-put%3A+The+results+of+an+inter-laboratory+study%5BTitle%5D)+AND+%22Microchem.+J%22%5BJournal%5D)">Curtain flow chromatography ('the infinite diameter column') with automated injection and high sample through-put: The results of an inter-laboratory study.</a> Microchem. J. 110 (0), 127-132 (2013).</li>
</ol>

This work was supported by UWS and ThermoFisher Scientific.

这项研究涉及CF色谱柱的实验室间分析测试效率和灵敏度方面的分析性能。在CF柱在节&#39;3的描述设置了双泵系统。双泵系统设定"来实现的40:60的流量比:在CF柱的入口（中心周）。的40:60（中心:外设）流量比通过设定各泵分别代表40％，总流量的60％，该值的流率实现的。在CF塔出口用以下在部分"中描述的方法调谐到为2.1毫米的ID的"虚拟"塔4。 CF出口流量的调整"。使用含有苯乙醚，丁基苯和戊基苯?...

近年来色谱柱技术的高效液相色谱法（HPLC），极大地推进;峰容量大大增加在很大程度上要归功于使用更小的颗粒尺寸和更高效的核壳粒子。因为分离通常是更有效的，因为峰现在更清晰，因此高1-8的流上作用已在敏感性的增加。 然而，径向床异质仍然在所有列的性能的一个限制因素，但是这不是一个新的故事，因为色谱已经知道这个多年。柱床有两个沿径向9-12异类，和沿列轴10,12-15。壁效应尤其是分离性能7,16-18损失的一个重要因素。 Shalliker和Ritchie 7最近审查柱床异质性方面，因此这不一定是discusseð这里进一步。虽然我只想说，在柱床堆积密度和变化的壁效应导致溶质插头的扭曲，使得乐队通过列塞类似于部分填充汤洗脱碗，而不是薄平固盘7通常是在基础教学文本描述。当实验进行，使得通过该床的溶质迁移可以被可视化的柱内的插头剖面是部分中空和带的拖尾部分主要是样品插头的壁组件。最终的结果是，它需要更多的板，以隔开比如果盘是固体和平面12,14,17将需要这些"部分空心&#39;插头。为了克服乐队扩大与壁效应和径向填充密度的变化有关的问题，被称为主动流动技术（AFT）柱技术的一种新形式的设计7,19。这种设计的目的是通过溶剂洗脱的沿着壁区域中的物理分离除去壁效果，从该流动相在柱19的径向中心区域洗脱。有两种主要类型的AFT列;并行分段流动（PSF）列和窗帘流量（CF）7列。由于该协议的目的是CF列的使用和优化，PSF列将不作进一步讨论。 幕流（CF） 幕流（CF）柱格式利用在两个入口和柱的出口AFT端部附件。 AFT端部附件包括位于一个多件内的环形熔块。玻璃料是由三部分组成:与端部的中心孔嵌合，与该端部接头的外周端口对准的多孔外部部分，和一个不渗透环对准的多孔径向中央部，其分离防止任何交叉的两个多孔部分玻璃料19的径向中心和外部区域之间-flow。 图1示出的AFT玻璃料的设计与图2示出在CF列格式。在此模式下操作的（CF）的样品被注入到入口配件的径向中央端口，而额外的移动相通过的径向中心区域通过所述入口到"帘"溶质的迁移的圆周口导入柱。因此，样品进入与具有流动相仅通过它的列的外部区域的列的径向中心区域的床。有研究表明，大约40:60的体积流率比（中央:周边端口），用于在入口4.6内径（ID）柱的端部配合的是最佳的6,7,16。在CF塔的AFT出口允许中枢和外周流量的调节其相对部分，并且可以被改变以几乎任何期望的拉提通过压力管理o。在CF列的优化可以显著改善塔技术的各种功能方面，例如分离效率或检测灵敏度。以这种方式，"无壁"，"无限直径"或"虚拟"列建立6,10,18,20。 CF列的目的是为了通过该柱主动管理样品的迁移，以防止样品在到达壁区域。因此，在出口到检测器中的溶质浓度为最大，当使用紫外线（UV）检测16，以及即使使用质谱检测6时更大的增加的比常规列格式更大约2.5倍的灵敏度。 CF列非常适合于低浓度的样品中，由于检测灵敏度增加。此外，联接到流率受限探测器，诸如质谱仪（MS）的6时它们是理想的。一个AFT柱在4.6毫米内径的格式，例如可以调整为标准2.1 mm内径格式列时在同一线速度操作时，通过调整中央流动退出到21％，提供的溶剂相同体积到检测器。同样，AFT列也可以调整，通过调整核心流程退出到43％，提供相同的容积负荷的检测器，3.0 mm内径柱。事实上任何"虚拟"列格式可以生产以满足需求分析6,18,22。在入口使用这些专门设计的端部附件和出口确保真正的无壁列成立。 有两种方法来设置溶剂输送系统入口的中心和周边港口:分流系统6和两个泵系统6,7 图3说明每个CF系统设置窗口中。 分流系统 一世呐分流系统（ 图3A）的泵的流量通向喷射器是使用零死体积T型件，其中流动相的一个流动流被连接到喷射器，其然后连接到分裂预喷射柱的入口端，嵌合的中央端口。流动相的第二流动流通过绕过喷射器，并连接到外围端口上的柱的入口。中流动的分割，在流动流百分比调节至40:60（中心:外围设备）的线连接到列之前， 即从喷射器到中心和泵外围。 双泵系统 在CF列需要在塔的入口端拟合两个流动流。根据高效液相色谱仪，分流设置的自动进样器/喷射器的类型，可能无法进行，所以CF然后可以通过两个泵（ 图3B 21）来实现。每个泵被分配，选择连接到中央或外围端口和流率被设定为表示流量为中央口的40％，为外围端口60％。例如，如果总流速1.0毫升分钟-1，中央泵流量被设为0.4毫升分钟-1和外围泵被设置为0.6毫升分钟-1。 其中操作方式的选择在很大程度上取决于该HPLC仪器和操作色谱模式。例如，在一些自动进样器中的样品装载位置和样品之间的压力的变化注入，可能会发生位置扰乱分流比，因此在这种情况下，一个双泵设置将被推荐为最佳的CF性能。无论设置溶剂输送系统选择用于CF柱的入口，在CF出口优化保持相同。在CF柱出口中央端口附连到与SMAL的紫外可见（UV-VIS）检测器免得体积可能管以减少柱后死体积的影响。因为，CF列模拟窄径柱，柱出口和检测器之间的死体积是不利的CF柱的分离性能。关键是要确保中央端口和UV-Vis检测以减少死体积的如带展宽，损失在效率和灵敏度的影响之间管容积的最小量。因此，使用窄口径管材（0.1毫米）的建议，以轻易让调整的压力不增加不适当的死体积。管也连接到所述外围端口，并指示浪费。当在CF柱的出口，所述分割比可以调节到适合分析者的目的的任何比率。当使用4.6 mm内径的CF，例如，它常常是方便设置的比率为要么43:57或21:79（中心:周），以模拟一个"虚拟"3.0毫米内径柱或2.1毫米内径柱，尊敬。这样的分离性能是容易长凳标记。分割比由称重流从连接到中央端口和流动离开该外围端口经过一段时间的检测器排出的量来测量。的百分比流量通过每个端口可以被确定，并且该比率可以通过改变管附着的长度或用具有不同的内部直径（ID）管进行调整。 此视频协议细节增强色谱性能CF塔的运行和优化程序。

<table><tbody><tr><td>HPLC instrument</td><td></td><td></td><td></td></tr><tr><td>Additional Pump</td><td></td><td></td><td>Required if 2 pump CF system set up is to be used.</td></tr><tr><td>Curtain Flow HPLC column</td><td>Thermo Fisher Scientific</td><td>Not Defined</td><td>Soon to be commercialized</td></tr><tr><td>Methanol</td><td>Any brand</td><td></td><td>HPLC Grade</td></tr><tr><td>PEEK tubing</td><td>Any brand</td><td></td><td>Various lengths and i.d.&amp;nbsp;</td></tr><tr><td>PEEK tube cutter</td><td>Any brand</td><td></td><td></td></tr><tr><td>Analytical Scale Balance</td><td>Any brand</td><td></td><td></td></tr><tr><td>Stop watch</td><td>Any brand</td><td></td><td></td></tr><tr><td>Eluent collection vessels</td><td>Any brand</td><td></td><td>1-2 ml sample vials can be used as eluent collection vessels</td></tr><tr><td>T-piece</td><td>Any brand</td><td></td><td></td></tr></tbody></table>

curtain flow column: optimization of efficiency and sensitivity

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially purpose built multiport end-fitting(s). Curtain Flow (CF) columns belong to the AFT suite of columns, specifically the CF column is designed so that the sample is injected into the radial central region of the bed and a curtain flow of mobile phase surrounding the injection of solute prevents the radial dispersion of the sample to the wall. The column functions as an 'infinite diameter' column. The purpose of the design is to overcome the radial heterogeneity of the column bed, and at the same time maximize the sample load into the radial central region of the column bed, which serves to increase detection sensitivity. The protocol described herein outlines the system and CF column set up and the tuning process for an optimized infinite diameter 'virtual' column.

AFT柱使用在多端口柱端部附件的专用料设计（ 图1）克服了柱床异质性和改善的分离性能的发展。上的CF层析柱（ 图2）的分离性能的实验室间研究用成立（ 图3B）作为在该协议23的第3节中描述的双泵系统进行的。三组分测试混合物通过一个"虚拟"2.1 mm内径其中CF塔的中心出口流量的21％被定向到检测器下进行分析。三组分测试?...

Watch this Scientific Journal Video about Curtain Flow Column: Optimization of Efficiency and Sensitivity at JoVE.com

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Here, we present a protocol for the operation and optimization of Active Flow Technology (AFT) column in Curtain flow (CF) mode for enhanced separation performance.

幕式柱:效率和灵敏度的优化

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially ...

curtain-flow-column-optimization-of-efficiency-and-sensitivity

Research

JoVE Journal

Chemistry

6.5K 次观看.  University of Western Sydney. Here, we present a protocol for the operation and optimization of Active Flow Technology (AFT) column in Curtain flow (CF) mode for enhanced separation performance. 

视频: Curtain Flow Column: Optimization of Efficiency and Sensitivity

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

科研

生物工程

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

Here, we present a protocol for the operation and tuning of parallel segmented flow chromatography columns to enable multiplexed detection.

调优并行分段流动列和启用多重检测

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms ...

化学

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

柱后衍生用反应流高效液相色谱柱

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

Optimization, Test and Diagnostics of Miniaturized Hall Thrusters

Miniaturized spacecraft and satellites require smart, highly efficient and durable low-thrust thrusters, capable of extended, reliable operation without attendance and adjustment. Thermochemical thrusters which utilize thermodynamic properties of gases as a means of acceleration have physical limitations on their exhaust gas velocity, resulting in low efficiency. Moreover, these engines demonstrate extremely low efficiency at small thrusts and may be unsuitable for continuously operating systems which provide real-time adaptive control of the spacecraft orientation, velocity and position. In contrast, electric propulsion systems which use electromagnetic fields to accelerate ionized gases (i.e., plasmas) do not have any physical limitation in terms of exhaust velocity, allowing virtually any mass efficiency and specific impulse. Low-thrust Hall thrusters have a lifetime of several thousand hours. Their discharge voltage ranges between 100 and 300 V, operating at a nominal power of &lt;1 kW. They vary from 20 to 100 mm in size. Large Hall thrusters can provide fractions of millinewton of thrust. Over the past few decades, there has been an increasing interest in small mass, low power, and high efficiency propulsion systems to drive satellites of 50-200 kg. In this work, we will demonstrate how to build, test, and optimize a small (30 mm) Hall thruster capable of propelling a small satellite weighing about 50 kg. We will show the thruster operating in a large space environment simulator, and describe how thrust is measured and electric parameters, including plasma characteristics, are collected and processed to assess key thruster parameters. We will also demonstrate how the thruster is optimized to make it one of the most efficient small thrusters ever built. We will also address challenges and opportunities presented by new thruster materials.

Here, we present a protocol to test and optimize space propulsion systems based on miniaturized Hall-type thrusters.

小型化霍尔推进器的优化、测试和诊断

Miniaturized spacecraft and satellites require smart, highly efficient and durable low-thrust thrusters, capable of extended, reliable operation without ...

工程学

Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach

Capillary ultra-high performance liquid chromatography (UHPLC) is currently a method of choice for the sample separation step in LC-MS-based proteomics. However, capillary columns are much less robust in comparison to their higher flow countertypes. Because of easy contamination and blocking, they often need replacement. That makes them a markedly expensive part of the total LC-MS analysis cost. In-house packing of UHPLC capillary columns saves a lot of money and allows customization. However, the standard packing procedure in the 100-bar pressure bomb works well only for HPLC columns but is too slow for UHPLC sorbents. Here we provide a description of an optimized FlashPack protocol applied to the same 100-bar pressure bomb setup. The method is based on packing from ultra-high sorbent concentration slurry and is developed for in-house manufacturing of UHPLC capillary columns of unlimited length in reasonable time.

Here we present a protocol for the optimized FlashPack capillary column packing procedure. Application of an optimized protocol to a common 100-bar pressure bomb setup allows 10-times faster packing and manufacturing of long ultra-high performance capillary columns.

使用FlashPack方法进行简单的内部超高性能毛细管柱制造

Capillary ultra-high performance liquid chromatography (UHPLC) is currently a method of choice for the sample separation step in LC-MS-based proteomics. ...

生物化学

Design and Optimization Strategies of a High-Performance Vented Box

This study aims to solve the problems of air flow chaos and poor performance in a vented box caused by the heterogeneous distribution of airflow through the design of the internal structure of the vented box with constant energy consumption. The final goal is to evenly distribute the airflow inside the vented box. Sensitivity analysis was performed for three structural parameters: the number of pipes, the number of holes in the middle pipe, and the number of each increment from the inside to the outside pipe. A total of 16 random array sets of three structural parameters with four levels were determined using the orthogonal experimental design. Commercial software was used for the construction of a 3D model for the selected experimental points, and this data was used to obtain the airflow velocities, which were then used to obtain the standard deviation of each experimental point. According to the range analysis, the combination of the three structural parameters was optimized. In other words, an efficient and economical optimization method considering the performance of the vented box was established, and it could be widely used to extend the storage time of fresh food.

Here, we present the range analysis method to optimize the sample points generated by an orthogonal experimental design to ensure that fresh food can be stored in a vented box for a long time by regulating the airflow pattern.

高性能通风箱的设计与优化策略

This study aims to solve the problems of air flow chaos and poor performance in a vented box caused by the heterogeneous distribution of airflow through ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

自动疏水层析蛋白质纯化用柱选择

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

生物学

High-Throughput Assays of Critical Thermal Limits in Insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

昆虫关键热极限的高通量测定

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...

Cross Cylindrical Flow: Measuring Pressure Distribution and Estimating Drag Coefficients

Source: David Guo, College of Engineering, Technology, and Aeronautics (CETA), Southern New Hampshire University (SNHU), Manchester, New Hampshire
The pressure distributions and drag estimations for cross cylindrical flow have been investigated for centuries. By ideal inviscid potential flow theory, the pressure distribution around a cylinder is vertically symmetric. The pressure distribution upstream and downstream of the cylinder is also symmetric, which results in a zero-net drag force. However, experimental results yield very different flow patterns, pressure distributions and drag coefficients. This is because the ideal inviscid potential theory assumes irrotational flow, meaning viscosity is not considered or taken into account when determining the flow pattern. This differs significantly from reality.
In this demonstration, a wind tunnel is utilized to generate a specified airspeed, and a cylinder with 24 ports of pressure is used to collect pressure distribution data. This demonstration illustrates how the pressure of a real fluid flowing around a circular cylinder differs from predicted results based on the potential flow of an idealized fluid. The drag coefficient will also be estimated and compared to the predicted value.

Cross Cylindrical Flow: Pressure Distribution &amp; Drag Coefficients

交叉圆柱流:测量压力分布和估计阻力系数

Source: David Guo, College of Engineering, Technology, and Aeronautics (CETA), Southern New Hampshire University (SNHU), Manchester, New Hampshire
The ...

教育

JoVE Science Education

航空工程

Efficiency of Liquid-liquid Extraction

Source: <a href="https://www.jove.com/author/Kerry+M._Dooley">Kerry M. Dooley</a> and Michael G. Benton, Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA
Liquid-liquid extraction (LLE) is a separation technique used instead of distillation when either: (a) the relative volatilities of the compounds to be separated are very similar; (b) one or more of the mixture components are temperature sensitive even near ambient conditions; (c) the distillation would require a very low pressure or a very high distillate/feed ratio.1The driving force for mass transfer is the difference in solubility of one material (the solute) in two other immiscible or partially miscible streams (the feed and the solvent). The feed and solvent streams are mixed and then separated, allowing the solute to transfer from the feed to the solvent. Normally, this process is repeated in successive stages using counter-current flow. The solute-rich solvent is called the extract as it leaves, and the solute-depleted feed is the raffinate. When there is a reasonable density difference between the feed and solvent streams, extraction can be accomplished using a vertical column, although in other cases a series of mixing and settling tanks may be used.
In this experiment, the operational goal is to extract isopropanol (IPA, ~10 - 15 wt. %, the solute) from a mixture of C8-to-C10 hydrocarbons using pure water as solvent. A York-Scheibel type (vertical mixers and coalescers, one each per physical stage) extraction column is available. Like most extractors, the overall efficiency (number theoretical stages/physical stages) of this column is quite low, especially in comparison to many distillation columns. The low efficiencies arise from both slow mass transfer (two liquid resistances instead of one as in distillation) and often also from maldistribution of the phases. The effect of agitator speed on both the solute recovery in the extract and the overall column efficiency will be evaluated.

Liquid-liquid Extraction and York-Scheibel Column Operation

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Source: Kerry M. Dooley and Michael G. Benton, Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA
Liquid-liquid extraction ...