Name: post column derivatization using reaction flow high performance liquid chromatography columns
Uploaded: 2016-04-26T13:00:00.000+00:00
Duration: 6 min 25 s
Description: Watch this Scientific Journal Video about Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns at JoVE.com

This work was supported by UWS and ThermoFisher Scientific. 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:此协议描述了特定于感兴趣的化学化合物的性质不同的试剂反应流柱后衍生的3种方法（RF-PCD）技术，每个。对于ROS的分析去一节"1. ROS的检测用DPPH•"，对于伯胺见的分析"2.检测使用荧光胺伯胺的"，而对酚类化合物的分析，去节"3 。酚的检测使用4 aminoantipyrene和铁氰化钾"，用超纯水（ 如 Milli-Q水）全程。 注意:射频柱的连接是在几乎相同的方式与主要差别是端部装配件的一个射频列的数目的常规HPLC柱来实现。用于连接标准HPLC柱的HPLC系统接头能够被用于一个射频柱连接到HPLC系统。 1. ROS的检测用DPPH• <ol><li> 设置HPLC仪器的 <ol><li>制备具有A线100％的水和B线100％甲醇作为流动相的高效液相色谱仪。吹扫泵按照制造商的要求。 </li><li>设置HPLC仪器组件和如在图4A中所示的附加 ​​衍生泵。 </li><li>设定UV-Vis检测在520纳米的波长进行分析。 </li></ol></li><li> 设置最多RF列<ol><li>射频柱的入口连接至HPLC仪器。 </li><li>连接0.13毫米ID管的15厘米长到出口的RF列的中心港。 </li><li>出口外设端口连接到采用0.13毫米内径管15厘米长的UV-Vis检测。 </li><li>的DPPH•泵线连接到外围端口上的射频柱的出口。 </li><li>阻止在使用柱塞射频列的出口的未使用的外设端口。 </li><li>带来的HPLC泵的流速到1ml分-1，在100％的线B - 100％甲醇。 </li><li>平衡与100％甲醇的流动相柱10分钟为4.6毫米内径×100毫米长柱。这个时间应该根据用户可采用其他列的尺寸来缩放。 </li></ol></li><li> 清除DPPH•试剂准备 <ol><li>制备在甲醇DPPH•的0.1mg / ml的溶液。 </l我&gt; <li>超声处理含有DPPH•试剂10分钟烧瓶中。 </li><li>盖瓶铝箔以防止暴露在光线下。 </li><li>吹扫DPPH•泵与制备DPPH•试剂按照制造商的要求。 </li></ol></li><li> 调谐RF柱出口 <ol><li>准确称取两个干净和干燥的船只。标签一艘船中央和其他外围。 </li><li>收集退出中央口插入标中部为1.0分钟，容器中的污水。 </li><li>重新称量中央口容器中，并从中央口计算流量的重量如下: 中央端口（克）的重量=中部港口船只（G）的最终重量 - 中环港容器的初始重量（g） </li><li>用于退出紫外可见是连接到RF列的外设端口和计算重量为外设端口容器中的流出物重复步骤1.4.2和1.4.3如下: 外设端口（克）的重量=外设端口船（G）的最终重量 - 外设端口容器的初始重量（g） </li><li>计算从中央和外围端口来如下流动的百分比: 中央％环港端口（克）=体重/×100（中央端口（G）+外设端口（克）重的重量） ％外设端口外设端口（克）=体重/×100（中央端口（G）+外设端口（克）重的重量） </li><li>确保中央流和外围流量之间的分割比为30:70（中央:周边）。如果中央流量为30％以上，通过添加0.13毫米ID管道的长度向中央端口的出口降低流动排出的量。如果中央流量低于30％从中央口减少0.13毫米管的长度。 </li><li>重复步骤1.4.1至1.4.6，直到30:70的分割比例（中央:外设）的实现。 </li><li>设置D的流量PPH•试剂泵0.5毫升分钟-1。 注意:设置与DPPH•试剂射频列现在准备进行分析。样品现在可以注入。 </li></ol></li><li> 运行后关机条件 <ol><li>一旦所有的样品被注射，表明运行完成，停止衍生试剂泵的流量。 </li><li>取下外设端口的DPPH•试剂泵线和塞住端口。 </li><li>平衡用流动相在其中是通过使流动相以1ml分通过塔传递-1 10分钟被存储在列中。 </li><li>停止对HPLC仪器流动相泵的流量。 </li><li>更换DPPH•试剂用甲醇和清除额外的泵。 注意:现在HPLC系统可以关闭。 </li></ol></li></ol> 2.使用Fluoresc伯胺的检测胺<ol><li> 流动相的制备 <ol><li>制备1升的10mM的醋酸铵溶液，将溶液的pH调节至9.0与5 M氢氧化铵稀释之前至体积。 </li><li>添加52.6毫升乙腈（ACN）的对乙酸铵缓冲以达到95:05（缓冲液:ACN）预混流动相。 </li></ol></li><li> 设置HPLC仪器的 <ol><li>与线A的预混合制备的缓冲液作为流动相制备HPLC仪器。吹扫泵按照制造商的要求。 </li><li>设置HPLC仪器组件和如在图4A中所示的附加 ​​衍生泵。 </li><li>附加脉冲阻尼器线圈衍生泵。 </li><li>设置荧光检测器（FLD）与475纳米390纳米，发射波长的激发波长。 </li></ol></li><li> 设置RF列 <ol><li>第连接射频列高效液相色谱仪电子入口。 </li><li>连接0.13毫米ID管的15厘米长到出口的RF列的中心港。 </li><li>射频柱的出口外设端口的连接采用0.13毫米内径的管道有15厘米长的FLD检测。 </li><li>衍生泵线连接到外围端口上的射频柱的出口。 </li><li>阻止在使用柱塞射频列的出口的未使用的外设端口。 </li><li>带来的HPLC泵的流速到1ml分-1为100％的线A -的10mM乙酸铵缓冲液pH 9中，用5％的ACN预混。 </li><li>平衡与100％的线路流动相柱10分钟为4.6毫米内径×100毫米长度列。这个时间可以根据用户可采用其他列的尺寸来缩放。 </li></ol></li><li> 荧光胺试剂的制备 <ol><li>赚100毫升0.1毫克毫升-1荧光胺试剂。 </li> &lt;利&gt;超声处理1分钟。 </li><li>用铝箔盖，防止暴露在光线下。 </li><li>吹扫与制备荧光胺试剂按照制造商的要求的试剂泵。 </li></ol></li><li> 调谐RF柱出口 <ol><li>准确称取两个干净和干燥的船只。标签一艘船中央和其他外围。 </li><li>收集退出中央口插入标中部为1.0分钟，容器中的污水。 </li><li>重新称量中央容器，并从中央口计算流量的权重作为在步骤1.4.3如下。 </li><li>重复步骤2.5.2至2.5.3用于退出是连接到RF列的外设端口和外围计算重量作为步骤1.4.4如下FLD的流出。 </li><li>计算从中央和外围端口来作为步骤1.4.5如下流的百分比。 </li><li>确保中央流和外围之间的分割比率流量是43:57（中央:外设）。如果中心流43％以上，通过添加0.13毫米ID管道的长度向中央端口的出口降低流动排出的量。如果中央流量低于43％，从中央口减少0.13毫米管的长度。 </li><li>重复步骤2.5.1至2.5.6，直到43:57分割比例（中央:外设）的实现。 </li><li>流动相泵的流速设置为0.7毫升分钟-1。 </li><li>设置衍生泵以0.1ml min至流动-1。 注意:设置与荧光试剂的RF列现在准备进行分析。样品现在可以注入。 </li></ol></li><li> 运行后关机条件 <ol><li>一旦所有的样品被注射，表明运行完成，停止衍生泵。 </li><li>除去从外设端口和塞子衍生泵线。 </li><li>在移动相平衡色谱柱它是通过使流动相以1ml分通过塔传递-1 10分钟被存储。 </li><li>停止流动相泵的流量。 </li><li>替换用乙腈荧光胺试剂和清除衍生泵。 注意:现在HPLC系统可以关闭。 </li></ol></li></ol> 3.酚的4 Aminoantipyrene和铁氰化钾的检测<ol><li> 流动相的制备 <ol><li>制备1升的的100mM乙酸铵溶液，将溶液的pH调节至9.0与5 M氢氧化铵稀释之前至体积。 </li><li>加52.6毫升甲醇的乙酸铵缓冲达到95的预混流动相:5（缓冲液:甲醇）。 </li></ol></li><li> 设置HPLC仪器的 <ol><li>与线A的预混合制备的缓冲液作为流动相制备HPLC仪器。吹扫泵按照制造商的氏要求。 </li><li>设置HPLC仪器组件和如图4B中所示的两个附加试剂泵。 </li><li>附加脉冲阻尼器线圈每一个试剂泵。 </li><li>设定UV-Vis检测在500nm的波长进行分析。 </li></ol></li><li> 设置RF列 <ol><li>射频柱的入口连接至HPLC仪器。 </li><li>连接0.13毫米ID管的15厘米长到出口的RF列的中心港。 </li><li>射频柱的出口外围端口连接到采用0.13毫米内径管15厘米长的UV-Vis检测。 </li><li>连接每个试剂（ 即 ，4-aminoantipyrene和铁氰化钾）泵线到RF射频列的出口的外设端口。 </li><li>带来的HPLC泵的流速到1ml分-1为100％的线A -的100mM乙酸铵缓冲液pH 9中，用5％甲醇预混合。 </li><li>平衡柱子W¯¯第i个10分钟为4.6毫米内径×100毫米长度列的100％线的移动相。这个时间可以根据用户可采用其他列的尺寸来缩放。 </li></ol></li><li> 4- aminoantipyrene试剂的制备 <ol><li>通过以下步骤3.1.1制备具有pH为9的醋酸铵缓冲液。 </li><li>重150毫克4- aminoantipyrene和在100毫升制备的乙酸铵缓冲液（pH 9）的溶解。 </li><li>超声处理1分钟。 </li><li>用铝箔盖，防止暴露在光线下。 </li><li>清除第一试剂泵与制备4- aminoantipyrene试剂按照制造商的要求。 </li></ol></li><li> 铁氰化钾的试剂制备 <ol><li>重150毫克的铁氰化钾和在按步骤3.1.1制备100毫升乙酸铵缓冲液（pH为9）溶解。 </li><li>超声处理1分钟。 </li><li>铝箔盖，防止暴露在光线下。 </li><li> PURGE中的第二试剂泵与制备铁氰化钾的试剂按照制造商的要求。 </li></ol></li><li> RF列的调整 <ol><li>准确称取两个干净和干燥的船只。标签一艘船中央和其他外围。 </li><li>收集退出中央口插入标中部为1.0分钟，容器中的污水。 </li><li>重新称量中央容器，并从中央口计算流量的权重作为在步骤1.4.3如下。 </li><li>重复步骤3.6.2至3.6.3用于退出紫外可见是连接到RF列的外设端口和外围计算重量作为步骤1.4.4如下的流出物。 </li><li>计算从中央和外围端口来作为步骤1.4.5如下流的百分比。 </li><li>确保中央流和外围流量之间的分割比率为50:50（中央:周边）。如果中央流量为50％以上，降低流通过添加0.13毫米ID管道的长度到出口中央口排出的量。如果中央流量低于50％时，从中央口减少0.13毫米管的长度。 </li><li>重复步骤3.6.1至3.6.6，直到50:50的分割比（中央:外设）的实现。 </li><li> 4 aminoantipyrene泵的流速设置为0.5毫升分钟-1。 </li><li>铁氰化钾泵的流速设置为0.25毫升分钟-1。 注:建立与双组分试剂射频列现在准备用于分析。样品现在可以注入。 </li></ol></li><li> 运行后关机条件 <ol><li>一旦所有的样品都被注入的运行已完成，表明运行完成，停止试剂泵。 </li><li>从外围端口移除试剂泵线并用15厘米的一块0.13毫米管替换它们。 </li><li>在WHI流动相平衡色谱柱通道它是通过使流动相以1ml分通过塔传递-1 10分钟被存储。 </li><li>停止对HPLC仪器流动相泵的流量。 </li><li>更换既对试剂试剂用甲醇泵和清除额外的泵根据制造商的要求。 注意:现在HPLC系统可以关闭。 </li></ol></li></ol>

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A. 96, 250-254 (1974).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+antioxidant+activity+of+ten+compounds+in+different+tea+samples+by+means+of+an+on-line+HPLC-DPPH+assay.">Evaluation of antioxidant activity of ten compounds in different tea samples by means of an on-line HPLC-DPPH assay.</a> Food Res Int. 53, 847-856 (2013).</li><li>Niu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+anti-oxidants+in+Flos+Chrysanthemi+by+HPLC-DAD-ESI/MS(n)+and+HPLC+coupled+with+a+post-column+derivatisation+system.">Identification of the anti-oxidants in Flos Chrysanthemi by HPLC-DAD-ESI/MS(n) and HPLC coupled with a post-column derivatisation system.</a> Phytochem Anal. 24, 59-68 (2013).</li><li>Raudonis, R., Bumblauskiene, L., Jakstas, V., Pukalskas, A., Janulis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+validation+of+post-column+assay+for+screening+of+radical+scavengers+in+herbal+raw+materials+and+herbal+preparations.">Optimization and validation of post-column assay for screening of radical scavengers in herbal raw materials and herbal preparations.</a> J. Chromatogr. A. 1217, 7690-7698 (2010).</li><li>Raudonis, R., Raudone, L., Jakstas, V., Janulis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+post-column+free+radical+scavenging+and+ferric+reducing+antioxidant+power+assays+for+screening+of+antioxidants+in+strawberries.">Comparative evaluation of post-column free radical scavenging and ferric reducing antioxidant power assays for screening of antioxidants in strawberries.</a> J. Chromatogr. A. 1233, 8-15 (2012).</li><li>Zakrzewski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Methimazole+in+Pharmaceutical+Preparations+using+an+HPLC+Method+Coupled+with+an+Iodine-Azide+Post-Column+Reaction.">Determination of Methimazole in Pharmaceutical Preparations using an HPLC Method Coupled with an Iodine-Azide Post-Column Reaction.</a> J. Liq. Chrom. Rel. Technol. 32, 383-398 (2008).</li><li>Zakrzewski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+reversed-phase+HPLC+method+with+post-column+iodine-azide+reaction+for+the+determination+of+thioguanine.">Development and validation of a reversed-phase HPLC method with post-column iodine-azide reaction for the determination of thioguanine.</a> J. Anal. Chem. 64, 1235-1241 (2009).</li><li>Gritti, F., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+measurements+of+the+true+column+efficiency+and+of+the+instrument+band+broadening+contributions+in+the+presence+of+a+chromatographic+column.">Accurate measurements of the true column efficiency and of the instrument band broadening contributions in the presence of a chromatographic column.</a> J. Chromatogr. A. 1327, 49-56 (2014).</li><li>Stewart, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post+cotumn+derivatization+methodology+in+high+performance+liquid+chromatography+(HPLC).">Post cotumn derivatization methodology in high performance liquid chromatography (HPLC).</a> Trends Anal. Chem. 1, 170-174 (1982).</li><li>Rigas, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-column+labeling+techniques+in+amino+acid+analysis+by+liquid+chromatography.">Post-column labeling techniques in amino acid analysis by liquid chromatography.</a> Anal. Bioanal. Chem. 405, 7957-7992 (2013).</li><li>Frei, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+Detectors+in+Modern+Liquid+Chromatography.">Reaction Detectors in Modern Liquid Chromatography.</a> Chromatographia. 15, 161-166 (1982).</li><li>Pravadil-Cekic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Reaction+Flow+Chromatography+for+the+Analysis+of+Amino+Acid:+Derivatisation+With+Fluorescamine+Reagent.">Using Reaction Flow Chromatography for the Analysis of Amino Acid: Derivatisation With Fluorescamine Reagent.</a> Microchem. J. , (2015).</li><li>Camenzuli, M., Ritchie, H. J., Dennis, G. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+segmented+flow+chromatography+columns+with+multiplexed+detection:+An+illustration+using+antioxidant+screening+of+natural+products.">Parallel segmented flow chromatography columns with multiplexed detection: An illustration using antioxidant screening of natural products.</a> Microchem. J. 110, 726-730 (2013).</li><li>Koleva, I. I., Niederlander, H. A. G., van Beek, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+On-Line+HPLC+Method+for+Detection+of+Radical+Scavenging+Compounds+in+Complex+Mixtures.">An On-Line HPLC Method for Detection of Radical Scavenging Compounds in Complex Mixtures.</a> Anal Chem. 72, 2323-2328 (2000).</li><li>Selim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Two-component+Post-column+Derivatisation+Method+Utilsing+Reaction+Flow+Chromatography.">A Two-component Post-column Derivatisation Method Utilsing Reaction Flow Chromatography.</a> Microchem. J. 116, 87-91 (2014).</li><li>Bigley, F. P., Grob, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Phenols+in+Water+and+Wastewater+by+Post-column+Reaction+Detection+High-performance+Liquid+Chromatography.">Determination of Phenols in Water and Wastewater by Post-column Reaction Detection High-performance Liquid Chromatography.</a> J. Chromatogr. A. 350, 407-416 (1985).</li><li>Camenzuli, M., Ritchie, H. J., Dennis, G. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+flow+chromatography+for+rapid+post+column+derivatisations:+The+analysis+of+antioxidants+in+natural+products.">Reaction flow chromatography for rapid post column derivatisations: The analysis of antioxidants in natural products.</a> J. Chromatogr. A. 1303, 62-65 (2013).</li></ol>

The authors have nothing to disclose.

RF-PCD允许与HPLC流出柱后衍生化试剂在不使用反应线圈的有效混合，减少频带加宽的影响，并提高分离性能。的RF-PCD方法也示于相对于检测方法的信号响应的改善。 Camenzuli 等 28是第一个报道的意式咖啡样品中DPPH•使用反应流列用于检测ROS的。他们的研究中所涉及的分析和射频条件的优化，以达到最大的性能，测试了一系列的DPPH•浓度与各种DPPH•试剂流速?...

再加上柱后衍生（PCD）的高效液相色谱（HPLC）是一个强大的工具，是在分析实验室解决的一些问题是有用的。它可用于检测与该套件可用1,2-探测器否则不可检测的化合物，增加目标分析物，这允许检测和定量3-5的下限或选择性的信号衍生的靶分析物，以避免基质效应6。常用的PCD反应包括胺类，如氨基酸的反应中，用邻- phthaladehyde 7-9，茚三酮9,10-或荧光胺11,12，活性氧的衍生（ROS）与2,2-二苯基1- picrylhydrazil自由基（DPPH•）13,14或2,2&#39;-连氮基-双（3-乙基苯并噻唑-6-磺酸（ABTS）15,16，以及使用碘化物叠氮化物试剂的衍生化磺丙ontaining化合物17,18。 有，但是，众多的缺点使用PCD反应的高效液相色谱系统6。在这些主要是添加衍生试剂（S）和所述检测器，其允许时间用于混合和发生8反应的点之间的使用反应线圈。这些反应环路通常具有500微升或更多，相对于HPLC系统19的其余部分的容积是显著的卷。使用这些高容量的反应循环中增加的峰加宽的结果相比，现在将未经反应环的存在下观察到的。这将导致在具有定量和检测的更高的限制更短，更宽的峰和负效应色谱分辨率。 图1和2突出，从加入各种柱后反应循环体积导致峰形状的恶化。这种分析用94％甲醇的流动相组合物和6％Milli-Q水进行。流动相的流速为1毫升/分钟，注射体积为20μl，并在分析波长为265纳米。从20微升至1000微升变化死体积的线圈插入柱并以模拟在PCD方法反应循环死体积的效应的检测器之间。这些环被从0.5mm内径的不锈钢管来制备。该实验由一个控制器（SCL-10AVP）的HPLC系统上进行，一个低压梯度阀（FCL-10ALVP），一个泵（LC-20AD）中，喷油器（SIL-10ADVP）和PDA检测器（ SPD-M10ADVP）。流动相通过脱气引入HPLC系统之前泵送。使用250毫米×4.6mm内径5微米柱进行分离。实验条件选择为典型的最近已经在文献中公开的PCD反应。 该最简单，最常见的柱后反应器设置被称为非分段的管状反应器，它实际上是一个长，细管，通过它可以使液体流出，反应可以发生。在这个系统中的峰变宽取决于不仅添加到系统中的死体积，而且还作为强调由饭岛等人 8的管的内径上。此外，线圈的几何形状起​​着观察品牌变宽的一部分。斯图尔特20指出，反应器的卷绕改变二次流剖面，导致更好的混合，这意味着死体积可以被最小化。据指出，峰宽用针织线圈21一 ​​个开放的管状时不显著。当峰加宽过大时，其它类型的反应器也可考虑20,22。这些可能包括床反应器或分段流反应器。这些反应器是反应慢了，否则会特别requir有用Ë大的反应循环。作为非分段的管状反应器是将PCD应用中最常用的类型的反应器，本文涉及这种类型的反应器装置的其余部分。 反应流程（RF）列的设计采用了多端口端部装配件，允许流动相，或者通过设在位于外柱的径向中心区域或三个端口的单个端口退出（或输入）该列列的壁区域（ 见图3）。这两个流使用含有由不可渗透的环是依次由延伸出到塔壁的外多孔玻璃料包围一个中央多孔玻璃料一个端部接头隔开。由于中央不透水环横流不是两个多孔区之间可能的。 在反应流层析，所述衍生化试剂（多个）被泵对流动相的流动方向成一个或总重量澳反应流柱的外部端口。柱洗脱剂通过自由外端口与外玻璃料的衍生试剂（多个）混合，并传递给检测器。反应流程可用于任一单一试剂衍生（1端口的衍生试剂，1端口到柱洗脱液传递给检测器和1个端口的阻塞状态）或双试剂系统（2个端口的衍生试剂和1端口通过柱洗脱剂的检测器）。从中央流的流动可以被用来检测未衍生柱洗脱液，有效地多路复用检测23，或传递给浪费。 一个主要的调整技术是可用时运行的RF-PCD色谱是中枢和外周流的比率。每个衍生的最佳比例取决于许多因素，如中央流是否将被检测或传递给浪费。因此，一旦最佳的比率已经确定，应当确保该正确流量比前正在执行每个运行来实现的。 业已发现，使用玻璃料的给列洗脱液流和射频PCD导致更有效的混合与通常采用零死体积T型件或低死体积的传统混合技术相比，衍生试剂混合W-片的两个流混合。这允许使用相对较小的反应的循环，或者在反应循环的甚至完全取消。反应回路尺寸的结果更清晰峰的减少相比传统的柱后衍生的方法。这意味着，尽管并非所有的柱洗脱液的衍生化，信噪比更大的信号被观察并且可以实现检测和定量的，因此下限。 反应流层析已经开发克服与PCD反应的适应性困难s至因造成的，需要采用大体积反应循环大柱后死体积现代HPLC柱和系统，特别是在致频带加宽效率的损失。相比于常规的PCD中的RF-PCD的更有效的混合过程意味着较小的反应环体积可以采用导致的增加观察到的分离效率。此外射频-PCD色谱同时显示增加的信号并进行比较，以产生相对于常规的PCD方法检测和定量的下限常规的PCD技术减少噪音。相比于常规的PCD方法的RF-PCD的另一个优点是，以监视来自RF列的中央端口以及从所述塔的外围区域洗脱衍生的流洗脱未衍生流的能力。 RF-PCD是一个相对较新的，但有前途的技术，该技术相对于传统的PCD方法显示许多优点。

<table><tbody><tr><td>HPLC instrument</td><td>Agilent</td><td>1290 Series HPLC</td><td></td></tr><tr><td>Additional Pump(s) for derivatization system</td><td>Shimadzu</td><td>LC-20A</td><td></td></tr><tr><td>RF colum</td><td></td><td></td><td>Non-commercial</td></tr><tr><td>PEEK tubing</td><td>Sigma Aldrich</td><td>Z227307</td><td></td></tr><tr><td>Column stoppers</td><td></td><td></td><td>Provided with column</td></tr><tr><td>PEEK tube cutter</td><td>Sigma Aldrich</td><td>Z290882</td><td></td></tr><tr><td>Analytical Scale Balance</td><td></td><td></td><td>4-point analytical balance</td></tr><tr><td>Stop watch</td><td></td><td></td><td>Non-Scientific equiptment</td></tr><tr><td>Eluent collection vials</td><td></td><td></td><td>Any Small vial with a flat bottom will do, &lt;em&gt;e.g.&lt;/em&gt;, HPLC vials</td></tr><tr><td>HPLC Vials</td><td></td><td></td><td>Will depend on instrument used</td></tr><tr><td>Vessels for mobile phase and derivatization solution(s)</td><td>Sigma Aldrich</td><td>Z232211</td><td></td></tr><tr><td>General Laboratory glassware</td><td></td><td></td><td>Volumetric Flasks, pippettes, &lt;em&gt;etc.&lt;/em&gt; Quantity and volumes will depend on sample preparation method.</td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>34860</td><td></td></tr><tr><td>DPPH</td><td>Sigma Aldrich</td><td>D9132</td><td></td></tr><tr><td>Ammonium Acetate</td><td>Sigma Aldrich</td><td>17836</td><td></td></tr><tr><td>Ammonia</td><td>Sigma Aldrich</td><td>320145</td><td>Corrosive</td></tr><tr><td>Acetonitrile</td><td>Sigma Aldrich</td><td>34998</td><td></td></tr><tr><td>Fluorescamine</td><td>Sigma Aldrich</td><td>F9015</td><td></td></tr><tr><td>4-aminoantipyrene&amp;nbsp;</td><td>Acros Organics BVBA</td><td>AC103151000</td><td></td></tr><tr><td>Potassium ferricyanide&amp;nbsp;</td><td>AnalaR</td><td>B10204-30</td><td></td></tr></tbody></table>

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.

这是适于通过射频PCD使用第一PCD方法是使用2,2-二苯基-1- picrylhydrazil自由基（DPPH•）24抗氧化剂的衍生。此反应通过Koleva 等人介绍了 25，并一直被广泛使用。检测依赖于DPPH•的脱色活性氧的存在下自由基，因此在观察到的吸光度的下降的抗氧化剂的结果存在。的DPPH•反应经常采用500微升或更13-15的大反应循环，但人们发现，使用RF-PCD?...

Watch this Scientific Journal Video about Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns at JoVE.com

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.

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 ...

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15.0K 次观看.  University of Western Sydney. A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

视频: Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

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 ...

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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.

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 ...

A Robust Method for Packing High Resolution C18 RP-nano-HPLC Columns

The high complexity prevalent in biological samples requires chromatographic separations with high sensitivity and resolution to be effectively analyzed. Here we introduce a robust, reproducible and inexpensive protocol for preparation of a nano-flow reversed phase high performance liquid chromatography (RP-HPLC) columns for on-line separation of analytical peptides before introduction into and detection by a mass-spectrometer in traditional bottom-up proteomics workflows. Depending on the goal of the experiment and the chemical properties of the analytes being separated, optimal column parameters may differ in their internal or outer diameters, length, particle size, pore size, chemistry of stationary phase particles, and the presence or absence of an integrated electrospray emitter at the tip. An in-house column packing system not only enables the rapid fabrication of columns with the desired properties but also dramatically reduces the cost of the process. The optimized protocol for packing a C18 AQ (aqueous) fused silica column discussed here is compatible with a wide range of liquid chromatographic instruments for achieving effective separation of analytes.

Here, we present and evaluate a protocol for making low cost reversed phase nano-flow liquid chromatography columns for peptide characterization using LC-MS/MS proteomic workflows.

The high complexity prevalent in biological samples requires chromatographic separations with high sensitivity and resolution to be effectively analyzed. ...

生物化学

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. ...

High-Performance Liquid Chromatography (HPLC)

Source: Dr. Paul Bower - Purdue University
High-performance liquid chromatography (HPLC) is an important analytical method commonly used to separate and quantify components of liquid samples. In this technique, a solution (first phase) is pumped through a column that contains a packing of small porous particles with a second phase bound to the surface. The different solubilities of the sample components in the two phases cause the components to move through the column with different average velocities, thus creating a separation of these components. The pumped solution is called the mobile phase, while the phase in the column is called the stationary phase.
There are several modes of liquid chromatography, depending upon the type of stationary and/or mobile phase employed. This experiment uses reversed-phase chromatography, where the stationary phase is non-polar, and the mobile phase is polar. The stationary phase to be employed is C18 hydrocarbon groups bonded to 3-&#181;m silica particles, while the mobile phase is an aqueous buffer with a polar organic modifier (acetonitrile) added to vary its eluting strength. In this form, the silica can be used for samples that are water-soluble, providing a broad range of applications. In this experiment, the mixtures of three components frequently found in diet soft drinks (namely caffeine, benzoate, and aspartame) are separated. Seven prepared solutions containing known amounts of the three species are used, and their chromatograms are then recorded.

Operation of High-Performance Liquid Chromatography HPLC

高性能液相色谱法 (HPLC)

Source: Dr. Paul Bower - Purdue University
High-performance liquid chromatography (HPLC) is an important analytical method commonly used to separate and ...

教育

JoVE Science Education

分析化学

Column Chromatography

Source: Laboratory of Dr. Jimmy Franco - Merrimack College
Column chromatography is one of the most useful techniques for purifying compounds. This technique utilizes a stationary phase, which is packed in a column, and a mobile phase that passes through the column. This technique exploits the differences in polarity between compounds, allowing the molecules to be facilely separated.1 The two most common stationary phases for column chromatography are silica gel (SiO2) and alumina (Al2O3), with the most commonly used mobile phases being organic solvents.2 The solvent(s) chosen for the mobile phase are dependent on the polarity of the molecules being purified. Typically more polar compounds require more polar solvents in order to facilitate the passage of the molecules through the stationary phase. Once the purification process has been completed&#160;the solvent can be removed from the collected fractions using a rotary evaporator to yield the isolated material.

Column Chromatography: Principle, Separation of Compounds from a Mixture

柱层析法

Source: Laboratory of Dr. Jimmy Franco - Merrimack College
Column chromatography is one of the most useful techniques for purifying compounds. This ...

有机化学

Principles Of Column Chromatography

The chromatography technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, to separate plant pigments using organic solvents. Further, in 1941, Archer John Porter Martin and R. L. M. Synge modified the technique by packing silica gel into a column. A mixture of amino acids was then separated on the packed column using chloroform and water mixture as the mobile phase. This was the first report on column chromatography. At present, column chromatography is a widely used technique for separating various types of compounds from a sample mixture.
Factors Influencing Efficient Separation of Proteins 
Various parameters like column material, packing, and operational conditions such as flow rates and temperature determine the efficiency of separation by column chromatography.
The choice of column material or matrix determines the extent of interaction with the sample. The matrix material must be tightly and uniformly packed in the column. Air bubbles, debris, large particles, and precipitates interfere with the uniform flow of the solvent through the column, affecting its separation efficiency. The column should also be free from particulate matter.
The sample injected into the column should be clear and free from aggregates that might clog the column, hindering solvent flow. The flow rate of solvent also affects the separation. Very high or very low flow rates of solvents result in inefficient separation of compounds and impure preparations. Very high rates may also disturb the column packing, affecting process efficiency. In addition, the composition of elution buffer is also an important factor. It should be non-corrosive and compatible with the sample and also the column material to prevent in situ precipitation or dissolution.
Another operational parameter, temperature, also plays an important role in the process. It decides the stability of the sample, column material, and solvent buffer. Also, a constant temperature throughout the column efficiently resolves compounds. After completing the separation process, the columns must be washed thoroughly by repeatedly passing a suitable solvent to avoid sample contamination in further runs. Occasionally, the solvent is passed in a column in the reverse direction to remove any clogged material.
Limitations
Though a very widely used technique, the method still has some limitations. It is a very time-consuming method as the flow rates need to be slower for better resolution of the compounds. Also, large quantities of highly pure solvents required in the mobile phase make the process expensive. This also raises the scaling-up cost when higher yields of pure compounds are needed.

柱色谱原理

The chromatography technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, to separate plant pigments using organic solvents. ...

细胞生物学

Analyzing Cells and Proteins

关键术语与概念

Types Of Column Chromatography

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.
Gel Filtration Chromatography 
When the protein&#39;s chemical nature is unknown, gel filtration chromatography is used for purification based on size. Matrices such as agarose, polyacrylamide, dextran derivatives, or silica beads are used for low-pressure systems, whereas polymeric resins are used for higher flow rates. The varying degree of cross-linking of these polymeric materials determines the pore size of the matrix, which decides the size range of proteins that can be separated.
Ion Exchange Chromatography
Ion-exchange chromatography can purify proteins based on their net charge at a particular pH determined by their isoelectric point or pI. The net charge on matrix resin is used to purify oppositely charged proteins by replacing the cations or anions from the matrix resin with the charged proteins. The resins are categorized based on the type of ion that is exchanged &#8212; positively charged proteins replace cations from cation-exchange resins, and vice versa. Strong ion exchangers with stability over a wide range of buffer pH include quaternary ammonium, sulfonate, and sulfopropyl resins. In contrast, weak ion exchangers that are effective only in a narrow pH range include diethyl aminoethyl (DEAE) or carboxymethyl (CM) resins.
Affinity Chromatography
Affinity chromatography involves immobilizing protein-specific antibodies or enzyme-specific substrates to the column material, usually agarose beads. For example, a column with the immobilized antibody of choice is prepared to separate specific antigens. When the mixed antigen sample is passed through the&#160; column, the target antigens bind to the immobilized antibodies and separate from the mixture. To separate recombinant proteins tagged with enzymes such as beta-galactosidase, the corresponding substrate Isopropyl-&#946;-D-thiogalactopyranoside (IPTG) is immobilized on the column to purify the enzyme.
High-pressure liquid chromatography (HPLC) and gas chromatography (GC) are the most commonly used column chromatography techniques. In HPLC, the solvent flows at high pressure through a tightly packed column that improves efficiency. Gas chromatography analyzes volatile compounds by converting them to their gaseous forms upon heating. The volatilized sample is pushed through the column by inert gasses such as nitrogen and helium.

柱色谱的类型

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating ...

High-Performance Liquid Chromatography: Introduction

High-performance liquid chromatography(HPLC), formerly referred to as High-pressure liquid chromatography, is a powerful technique used to separate, identify, and quantify components in complex mixtures. The term &#34;high pressure&#34; refers to using high pressure to push the liquid mobile phase through the tightly packed columns.
In HPLC, two phases play a critical role in the separation process:
<ol>
	<li>Mobile Phase: This is a liquid solvent that flows through the system, carrying the sample with it. The solvent is chosen based on its polarity to optimize the interaction with the analytes and the stationary phase. The &#34;polarity index&#34; serves as a guide to selecting the suitable solvent for separation.</li>
	<li>Stationary Phase: The stationary phase is a solid material, often silica-based, packed within the column. In reverse-phase HPLC, the stationary phase is typically derivatized silica, such as octadecyl (C18) silica. The small particle size of the packed material creates a high surface area, enhancing interactions with the analytes. To reduce undesirable interactions, such as those with unreacted silanol groups, the stationary phase is often end-capped with reagents like trimethylchlorosilane</li>
</ol>
Separation occurs as the analytes interact differently with both the mobile and stationary phases, depending on their chemical properties, which leads to differential migration through the column.
Sometimes, solute molecules bind irreversibly to the stationary phase, clogging the analytical column and decreasing its performance. To prevent this, a guard column is placed before the analytical column. A precolumn called a scavenger column may also be placed between the mobile phase reservoir and the injector. This ensures that the solvent enters the analytical column, which is already saturated with silica from the scavenger column, preventing unwanted reactions and extending the lifetime of the analytical column.
After the separation, analytes are detected by various detectors, depending on the characteristics of the compounds. The results are visualized as chromatograms, with peaks corresponding to the different components of the mixture. This provides both qualitative and quantitative data about the sample.

高效液相色谱:简介

High-performance liquid chromatography(HPLC), formerly referred to as High-pressure liquid chromatography, is a powerful technique used to separate, ...

柱后衍生用反应流高效液相色谱柱

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A Robust Method for Packing High Resolution C18 RP-nano-HPLC Columns

使用FlashPack方法进行简单的内部超高性能毛细管柱制造

高性能液相色谱法 (HPLC)

柱层析法

柱色谱原理

柱色谱的类型

高效液相色谱:简介

高效液相色谱:简介