二维液相色谱(SCX/RP)系统的构建及对珠蛋白水解产物的研究

以十通阀为切换接口, 构建了SCX/RP常规柱二维液相色谱系统, 并以珠蛋白水解产物的分析对其加以评价. 样品首先由第一维阳离子交换色谱(Hypersil SCX, 100 mm×4.6 mm I.D.)在pH 4.0的磷酸盐缓冲体系中分离, 洗脱产物进入切换接口, 样品组分被富集在捕集柱(Hypersil BDS C18, 15 mm×4.6 mm I.D.)中, 进一步脱盐后被导入第二维反相色谱(Hypersil BDS C18, 250 mm×4.6 mm I.D.)分离分析. 阳离子交换色谱采用逐步增加盐浓度的12步台阶等度间断方式洗脱, 每次将洗脱产物捕集在捕集柱中进而由反相色谱分析, 实现对第一维洗脱产物的切割转移及第二维分析. 与一维色谱相比, 二维液相色谱系统的分辨率、峰容量也得到提高, 系统峰容量达到2280.     

全二维液相色谱(IEC/RP)的构建与评价

以IEC/RP模式构建了二维液相色谱系统,采用平行交替柱捕集分析的2位十通切换阀作接口,第一维洗脱产物按10:1的分流比分流后,得到IEC和RP切换谱图.以5个标准蛋白混合物的分离评价该系统,在单独一维模式中不能分离的样品在全二维液相色谱中得到了很好的分离.     

IEX/RP二维液相色谱中弱保留组分收集模式的构建及系统评价

以十通阀和捕集柱接口形式,构建了弱阴离子交换/反相(WAX/RP)二维液相色谱分离系统.通过将第一维离子交换色谱分析中的前部集中洗脱出的弱保留组分收集后单独分析,剩余样品进一步采用二维液相色谱分析,可以有效避免第二维色谱柱对特殊样品局部集中流出导致的第二维分离超柱容量问题,提高了系统的整体分离能力.使用4种蛋白胰蛋白酶酶解后的多肽样品评价该系统,在不分流的系统中共检测到115个峰.对第一维分离的前15 min分流后得到的组分单独分析,共识别出58个峰,后续的二维分离中共得到78个峰.2种方法相比,第二种方法检测峰数增加了21个,一些低丰度的组分在弱保留组分收集后被识别.     

高温二维液相色谱系统的构建及其评价

在二维液相色谱中, 第二维的分离速度是制约其发展的重要因素. 升高色谱柱温度可以有效降低流动相粘度, 加快溶质在两相间的传质速率, 有效加快分析速度. 以离子交换色谱法(WAX)为第一维分离模式和反相色谱法(RP)为第二维分离模式, 十通阀和两个捕集柱为接口, 通过将第二维色谱柱温度升高到80 ℃和提高流量到3 mL/min, 构建了高温WAX/RP二维液相色谱系统. 以4种标准蛋白的酶解物为样品评价系统的分离性能, 第一维共有33个馏分被捕集并导入到第二维分析, 高温二维液相色谱系统识别出187个色谱峰.     

高温正相色谱/反相色谱(HTNPLC/RPLC)二维联用系统的构建与应用

为解决含水正相色谱与反相色谱联用过程中的谱带展宽问题,以高温正相色谱(HTNPLC)为第一维,室温反相色谱(RPLC)为第二维,分别建立了切割模式的二维液相色谱(HTNPLC-μRPLC)和全二维液相色谱(HTNPLC×RPLC)系统.样品首先在第一维(CN色谱柱)进行正相分离,第一维洗脱产物选择性地或者交替存储在十通阀上的定量环中,然后切换十通阀将定量环内存储的样品组分转移到第二维(C18色谱柱)进行反相分离.该系统降低了第一维切割组分在第二维柱头的扩散,提高了分离度以及分离效率.采用多环芳烃混合物和天然植物甘草提取液对该二维液相色谱系统进行了评价.     

全二维液相色谱的初步构建及其在山羊血清分离中的应用

以GFC/RP模式构建全二维液相色谱系统,第一维凝胶过滤色谱柱使用ShodexProteinKW 802. 5(300mm×8mmi.d. ),以0. 2mol/LNaH2PO4 (pH7. 0)的流动相在0. 15mL/min的流速下等度洗脱,第二维反相色谱柱使用HypersilBDSC18 (35mm×4. 6mmi.d. ),在3mL/min的流速下梯度洗脱。采用平行柱交替分析的形式作切换接口, 2. 5min切换一次,两个反相柱交替富集、分析第一维洗脱产物。以5个标准蛋白混合物的分离评价该系统,在单独一维模式中不能分离的样品在全二维液相色谱中得到了较好的分离,二维系统的总峰容量为225。与一维色谱相比,系统的总峰容量、分辨率得到较大提高。并用于山羊血清的纯化分析,对一维分离中的重合谱峰进行验证,对制备纯化有一定的实际意义。     

六味地黄丸组分的二维液相色谱分离

以1个常规六通阀直接连接两支常规尺寸的色谱柱(250 mm×4.6 mm i.d.),构建简单的SCX/RP在线二维液相色谱系统,对中成药六味地黄丸组分进行了优化分离。样品经过第一维阳离子交换色谱(Hypersil SCX),洗脱产物分离后通过六通阀直接富集到反相分析柱(C18)顶端,被转移到第二维色谱柱上继续进行分离。经过11步不连续的线性梯度洗脱,二维分离系统出峰数量达到550多个,峰容量达到2266。构建的二维液相色谱系统结构简单,与一维色谱相比,具有分辨率高、峰容量大的特点。     

真空溶剂蒸发全二维液相色谱接口及其应用

以正相色谱(NPLC)为第一维,反相色谱(RPLC)为第二维,建立了真空溶剂蒸发接口的全二维液相色谱系统(VEI-C2DLC)。样品首先在第一维(CN色谱柱)进行正相分离,第一维洗脱产物被交替存储到十通阀上的两个定量环中,与此同时对切割到定量环内的第一维组分进行在线真空蒸发,被分析样品组分保留在定量环内壁内,而溶剂被蒸发除去。十通阀切换后保留在该定量环内的样品组分被洗脱到第二维进行反相分离,如此反复循环使第一维组分完全转移到第二维。采用标准样品和天然植物蛇床子提取液对该全二维液相系统进行了评价。     

六昧地黄丸组分的二维液相色谱分离

以1个常规六通阀直接连接两支常规尺寸的色谱柱(250 mm×4.6 mm i.d.),构建简单的SCX/RP在线二维液相色谱系统,对中成药六味地黄丸组分进行了优化分离.样品经过第一维阳离子交换色谱(Hypersil SCX),洗脱产物分离后通过六通阀直接富集到反相分析柱(C18)顶端,被转移到第二维色谱柱上继续进行分离.经过11步不连续的线性梯度洗脱,二维分离系统出峰数量达到550多个,峰容量达到2266.构建的二维液相色谱系统结构简单,与一维色谱相比,具有分辨率高、峰容量大的特点.     

全二维液相色谱(NPLC×RPLC)接口及其应用

用内径为0.53 mm的填充毛细管正相液相色谱为第一维, 用4.6 mm(i.d.)×50 mm RP-18e整体柱反相色谱为第二维, 建立了定量环-阀切换接口的全二维液相色谱系统(NPLC×RPLC). 第一维色谱分离洗脱出的组分交替存储在十通阀上的两个定量环中, 同时定量环中前一个组分被转移到第二维进行反相分离. 因为第一维的流动相流量仅是第二维的1/500, 自然解决了流动相兼容问题. 采用芳香族化合物的混合物和中药丹参正己烷提取液对该全二维液相系统的分离能力进行了评价.     

Comprehensive two-dimensional capillary supercritical fluid chromatography in stop-flow mode with synchronized pressure programming

A comprehensive two-dimensional capillary supercritical fluid chromatography method was developed. The interface consisted of a ten-port valve, a capillary trap and two fused silica restrictors. The primary column was operated in stop-flow mode: the flow in the primary column was stopped during the separation of the second dimension. The pressure of the system was controlled with a single pump. The pressure program was synchronized with the sampling: the pressure was only ramped up during the sampling time, when the primary column effluent was transferred from the first dimension to the trap, and was maintained constant during the second-dimension separation. All of the operations were automated using in-house software. The separation characteristics of the present system can be readily regulated by changing the size of the restrictors and/or the programmed pressure rate. The use of synchronized pressure programming allowed the sampling duration and/or the second-dimension separation time (and therefore, the total analysis time) to be changed without affecting the separation pattern. Widely different selectivities were attained depending on the combination of the three columns with different polarities (such as the nonpolar DB-1, the medium-polarity DB-17 and the polar DB-WAX columns) used. The present system afforded improved separation and identification capabilities for analytes in complex mixtures.     

凝胶过滤/离子交换全二维液相色谱系统的构建与评价

基于蛋白质的尺寸及带电性质,将凝胶过滤色谱(GFC)与离子交换色谱(IEC)两种分离模式结合,采用双捕集柱接口构建了GFC/2×IEC二维液相色谱(2-D LC)分离系统,同时考虑离子交换色谱分离蛋白质对等电点范围的限制,进一步结合中心切割平行柱的方法实现对蛋白质的全二维分离。为与后续蛋白质在线酶解、多肽分离及质谱鉴定匹配,系统中采用常规柱以保证蛋白质质谱鉴定对样品量的要求,3种常规分离柱分别选用凝胶过滤色谱柱TSK-GEL G3000SW_(XL)(300 mm×7.8 mm,5μm)、强阴离子交换色谱柱Hypersil SAX(100 mm×4.6 mm,10μm)和强阳离子交换色谱柱Hypersil SCX(100 mm×4.6 mm,10μm)。最终以酵母细胞蛋白质提取液为样品,对构建的二维系统加以评价,在总蛋白质浓度13.5 mg/mL、上样体积100μL的条件下,将第一维分离等时间切割17次,并将切割馏分全部导入第二维继续分离,二维系统在148 min内获得的总峰容量达到884。说明所构建的系统可以用于蛋白质的在线全二维分离。     

Two-dimensional separation system of coupling capillary liquid chromatography to capillary electrophoresis for analysis of Escherichia coli metabolites

A two-dimensional (2-D) separation system of coupling chromatography to electrophoresis was developed for profiling Escherichia coli metabolites. Capillary liquid chromatography (LC) with a monolithic silica-octadecyl silica column (500 x 0.2 mm ID) was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension. Field-enhanced stacking was selectively employed as a concentration strategy to interface the two dimensions, which proved to be beneficial for the detection of metabolites. An artificial sample containing 118 standards, some of which lack chromophores or have weak UV absorbance, was used to optimize the 2-D separation system. Under the optimum conditions, 63 components in the artificial sample having absorbance at 254 nm could be well resolved and detected. The utility of the system was demonstrated by comprehensive analysis of E. coli metabolites. Comparing with the previous 2-D separation system we published in Anal. Chem. 2004, 76, 1419-1428, using a longer monolithic column in the first dimension improved the separation efficiency and offered the possibility of increasing the injection volume without compromising the separation efficiency. In the second dimension, field-enhanced stacking was used to improve the concentration sensitivity of the metabolites, and more metabolites in E. coli cell extract were detected and identified using the developed 2-D separation system. In addition, preliminary investigation for future CE-mass spectrometry coupling was also made in the study by using volatile buffers in the capillary LC and CE techniques.     

Two-dimensional capillary liquid chromatography: pH gradient ion exchange and reversed phase chromatography for rapid separation of proteins

In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4,000 A) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pH-fractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.     

A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatography x liquid chromatography

A novel approach for the selection of the operational parameters (linear velocity, column length) for a comprehensive 2D-LC system is discussed. Starting point for the calculations is a given second dimension ((2)D) separation and a desired peak capacity for the 2D system. Using the theory developed here the optimum settings for the first dimension ((1)D) column can be derived. Theory clearly indicates that the choice of the (1)D conditions is basically limited to just one set of column lengths and linear velocities. The new method is tested on a comprehensive two-dimensional liquid chromatography system which uses size-exclusion chromatography (SEC) followed by reversed phase liquid chromatography (RPLC). A novel LC/LC interface, using a six-port valve rather than storage loops, joins the two chromatographic dimensions. From a theoretical comparison of continuous low flow and stop-flow operation the latter method was found to be an attractive mode of interfacing. The common idea that stop-flow operation results in additional band broadening is shown to be incorrect. The new interface design operated in the stop-flow mode permits the use of conventional analytical diameter HPLC columns, 7.8mm for SEC and 4.6mm for RPLC. The reversed phase chromatography utilizes a monolithic C-18 modified silica column, which produces fast and efficient analyses. As test samples complex mixtures of peptides were analyzed.     

New zwitterionic polymethacrylate monolithic columns for one‐ and two‐dimensional microliquid chromatography

We prepared 0.53 and 0.32 mm id monolithic microcolumns by in situ copolymerization of a zwitterionic sulfobetaine functional monomer with bisphenol A glycerolate dimethacrylate (BIGDMA) and dioxyethylene dimetacrylate crosslinkers. The columns show a dual retention mechanism (hydrophilic‐interaction mode) in acetonitrile‐rich mobile phases and RP in highly aqueous mobile phases. The new 0.53 mm id columns provided excellent reproducibility, retention, and separation selectivity for phenolic acids and flavonoids. The new zwitterionic monolithic columns are highly orthogonal, with respect to alkyl silica stationary phases, not only in the hydrophilic‐interaction mode but also in the RP mode. The optimized monolithic zwitterionic microcolumn of 0.53 mm id was employed in the first dimension, either in the aqueous normal‐phase or in the RP mode, coupled with a short nonpolar core‐shell column in the second dimension, for comprehensive 2D LC separations of phenolic and flavonoid compounds. When the 2D setup with the sulfobetaine–BIGDMA column was used for repeated sample analysis, with alternating gradients of decreasing (hydrophilic‐interaction mode), and increasing (RP mode) concentration of acetonitrile on the sulfobetaine–BIGDMA column in the first dimension, useful complementary information on the sample could be obtained.     

Time-resolved cryogenic modulation for targeted multidimensional capillary gas chromatography analysis

Multidimensional gas chromatography (MDGC) is performed in a new manner, described in this paper. The method incorporates two directly coupled columns and employs a longitudinally modulated cryogenic trap located between the columns. No heartcutting process is used, but rather a method better termed selected zone compression pulsing is used. Compared with normal MDGC, where primary column effluent has to be temporarily diverted either to a monitor detector or to the second dimension column, the new procedure in its simplest mode passes all of the first column effluent to the second column. It is simply the times at which the modulation of the trap is performed that determines which target solutes will be selected for enhanced separation. This approach allows almost instantaneous separation of selected zones on the second column, and has the potential to significantly simplify the MDGC method. Since data are presented in a time-response format, and do not require transformation as previously described for comprehensive GC when using the longitudinal modulator, quantitation and report generation are essentially the same as in any GC method and data system. Advantages also include significant sensitivity improvement. By using cryofocussing, and benefiting from the zone compression effects along with fast GC conditions on the second dimension, new possibilities for MDGC can be realised. The method is demonstrated by using a mixture of semi-volatile aromatic hydrocarbons.     

Comparison of separation power of ultra performance liquid chromatography and comprehensive two-dimensional liquid chromatography in the separation of phenolic compounds in beverages

In this study, 1-D and 2-D liquid chromatographic systems, namely, conventional HPLC, UPLC, HPLC x HPLC and HPLC x UPLC systems were developed and evaluated for the separation of phenolic acids in wine and juices. In the LC x LC studies, the first dimension separation was based on RPLC and the second dimension was performed with ion-pair chromatography. Three different columns, namely two short columns packed with either 2.5 or 1.7 microm particles and a monolithic column, were tested for the fast second dimension separation. The best results were obtained when the monolithic column was applied for the second dimension separation. The peak capacities for comprehensive 2-D systems varied from 330 to 616.