Effect of first dimension phase selectivity in online comprehensive two dimensional liquid chromatography (LC×LC)

In this study, we examined the effect of first dimension column selectivity in reversed phase (RP) online comprehensive two dimensional liquid chromatography (LC×LC). The second dimension was always a carbon clad metal oxide reversed phase material. The hydrophobic subtraction model (HSM) and the related phase selective triangles were used to guide the selection of six different RP first dimension columns. Various kinds of samples were investigated and thus two different elution conditions were needed to cause full elution from the first dimension columns. We compared LC×LC chromatograms, contours plots, and fcoverage plots by measuring peak capacities, peak numbers, relative spatial coverage, correlation values, etc. The major finding of this study is that the carbon phase due to its rather different selectivity from other reversed phases is reasonably orthogonal to a variety of common types of bonded reversed phases. Thus quite surprisingly the six different first dimension stationary phases all showed generally similar separation patterns when paired to the second dimension carbon phase. This result greatly simplifies the task of choosing the correct pair of phases for RP×RP.     

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.     

2D LC Separation and Determination of Bradykinin in Rat Muscle Tissue Dialysate with On-Line SPE-HILIC-SPE-RP-MS

A 2D liquid chromatography (LC) system using hydrophilic interaction chromatography (HILIC) and reversed phase columns has been employed for comprehensive (LC × LC) separation of rat muscle tissue micro-dialysate. Incorporation of an on-line reverse-phase solid phase extraction (SPE) enrichment column in front of the first dimension enabled aqueous samples with high salt concentrations to be injected directly without compromising the chromatographic performance of the HILIC column. Since the SPE enrichment column allowed injection of large sample volumes (e.g. 450 μL), a capillary HILIC column (inner diameter 0.3 mm) could be employed instead of a larger column which is often used in the first dimension to load sufficient amounts of sample. The two chromatographic dimensions were connected using a column selector system with 18, 1.0 mm I.D. C₁₈ “transition” SPE columns. A PLRP C₁₈ column was used in the second dimension. The 2D LC system’s performance was evaluated with a tryptic digest mixture of three model proteins. Good trapping accuracy (HILIC→transition SPE→RP recovery >95%) and repeatability (within-and between day retention time RSDs of first and second dimension chromatography >1%) was achieved. A dialysis sample of rat muscle tissue was separated with the 2D system, revealing complexity and large differences in concentrations of the various compounds present, factors which could potentially interfere with the quantification and monitoring of two target analytes, arg-bradykinin and bradykinin. Subsequently, “Heart-cut” 2D LC-electrospray–mass spectrometry (ESI–MS) with post-column on-line standard injection was employed to monitor arg-bradykinin and bradykinin levels as a function of various muscle conditions. The method’s quantification precision was RSD = 3.4% for bradykinin.     

Preparative isolation of novel antioxidant flavonoids of alfalfa by stop-and-go counter-current chromatography and following on-line liquid chromatography desalination

In this work, we have established a new stop-and-go two-dimensional chromatography coupling of counter-current chromatography and liquid chromatography (2D CCC × LC) for the preparative separation of two novel antioxidant flavonoids from the extract of alfalfa (Medicago sativa L.). The CCC column has been used as the first dimension to purify the target flavonoids using a solvent system of isopropanol and 20% sodium chloride aqueous solution (1:1, v/v) with the stop-and-go flow technique, and the LC column packed with macroporous resin has been employed as the second dimension for on-line absorption, desalination and desorption of the targeting effluents purified from the first CCC dimension. As a result, two novel flavonoids, 6,8-dihydroxy-flavone-7-O-β-D-glucuronide (15.3 mg) and 6-methoxy-8-hydroxy-flavone-7-O-β-D-glucuronide (13.7 mg), have been isolated from 126.8 mg of crude sample pre-enriched by macroporous resin column. Their structures have been identified by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) and one- and two-dimensional nuclear magnetic resonance spectra (1D and 2D NMR). Further antioxidant assays showed that the first component possess a strong antioxidant activity. All the results demonstrated that the stop-and-go 2D CCC × LC method is very efficient for the separation of flavonoids of alfalfa and it can also be applied to isolate other comprehensive multi-component natural products.     

The acetonitrile shortage: Is reversed HILIC with water an alternative for the analysis of highly polar ionizable solutes?

In hydrophilic interaction chromatography (HILIC), best results are obtained with high concentrations of ACN. In the framework of green chromatography and the present shortage and very high price of this hazardous solvent, reversing the stationary phase to apolar and the mobile phase to aqueous can be of interest for several applications. The features of the aqueous RP technique called per aqueous LC (PALC) are illustrated with the analysis of catecholamines, nucleobases, acids, and amino acids. The ca. three-fold higher viscosity of water compared to ACN has consequences on the shape of the Van Deemter plot. For dopamine (N = 26.450 on a 25 cm×4.6 mm id, 5 μm bare silica column), a reduced plate height of 1.9 at an u_opt of 0.3 mm/s was calculated. The plate number, however, strongly depends on pH and ionic strength. As in RP separations, retention is shortened by adding an organic modifier. In the framework of green chromatography, the biodegradable ethanol was used. On the other hand, retention increased by lengthening the carbon chain of ion-pair reagents supporting the RP mechanism as well.     

Ion-pair Reversed-phase×Low-pH Reversed-phase Two-dimensional Liquid Chromatography for In-depth Proteomic Profiling

High-resolution liquid chromatography separation is essential to in-depth proteomic profiling of complex biological samples. Herein, we established an ion-pair reversed-phase×reversed-phase two-dimensional liquid chromatography (IPRP×RP 2DLC) strategy for comprehensive proteomic analysis. Both RPLC separation dimensions were performed at low pH, with trifluoroacetic acid(TFA) and formic acid(FA) as mobile phase addictive, respectively. As the good separation resolution offered by ion-pairing effect of TFA, the fractionation efficiency was greatly improved with 74.0% peptides identified in just one fraction. Comparing with conventional high pH RP fractionation, the overall separation rate of IPRP was about 1.6 times that of high-pH RP, which increased the number of identified peptides by 21%. Further, 2169 proteins and 8540 peptides were confidently identified from crude serum sample by our IPRP×RP 2DLC strategy, exhibiting great potential in clinical proteomics in the future.     

Determination of selectivity of HPLC systems by correspondence factor analysis

Correspondence factor analysis （CFA） was employed to study the selectivity of 14 HPLC systems. The tested LC systems were classified as reversed-phase （RP）, ion-exchange （IE） and hydrophilic interaction chromatography （HILIC） modes. It was found that the retentions of the hydrophilic solutes on HILIC column were significantly influenced by the second-order effects besides their hydrophilic properties. Organic modifiers and residue silanol groups on silica surface both participated in retention. HypersilTM amino column performed separation in the HILIC mode at appropriate conditions, and its retention mechanism was more similar to that of HILIC silica column than that of HILIC column coating poly（aspartamide） groups.     

二维液相色谱接口的改进及其在蛋白质组学研究中的应用

随着蛋白质组学、本草物质组学等组学概念的提出,所需分析的样品的成分越来越复杂,因此具有强大分离能力的多维液相色谱技术受到人们越来越多的关注。二维液相色谱中第二维的分离性能和速度是整个分离系统性能的关键。基于捕集柱模式,我们采用经特殊设计的流路系统,使得双捕集柱型接口具有预分离的功能。样品从第一维流出以后被富集在捕集柱1的柱头,经过脱盐后,正冲捕集柱,捕集柱1与第二维色谱柱联用对富集的样品进行分离,增加了第二维分离效率。当捕集柱上的样品全部被洗脱到第二维色谱柱上时,捕集柱2已经完成对第一维洗脱液中样品的捕集和脱盐,此时将阀进行切换,捕集柱2与第二维色谱柱直接相连进行洗脱。循环切换捕集柱1和捕集柱2,维持较高的阀切换频率,实现了第二维色谱柱的连续洗脱。因此保证了第二维分离具有较快速度,同时具有较高的分离效率。使用35 mm长捕集柱和十通阀为接口,以弱阴离子交换(WAX)色谱为第一维分离模式,以反相(RP)色谱为第二维分离模式,构建了WAX-RP二维液相色谱系统(2D-LC system)。以小鼠血清为样品对系统进行了初步评价。色谱流出曲线出现了明显的界面现象,这是由于捕集柱流动相中含有的较多盐分流出时的背景吸收造成的。同时,由于界面两侧的流动相黏度不同产生了黏性指进(VF)现象。当第二维色谱柱长度为50 mm时,理论上可将第二维分离效能提高70%。该接口可以应用于多种二维液相色谱模式,适用于蛋白质组学和本草物质组学研究中对于复杂样品的分离分析。     

Optimization of two-dimensional gradient liquid chromatography separations

An almost orthogonal comprehensive two-dimensional liquid chromatography was developed for the separation of phenolic and flavone natural antioxidants by using combinations of a polyethylene glycol silica micro-column in the first dimension and a porous-shell fused-core C18 column in the second dimension, both in the reversed-phase mode. System orthogonality was improved using parallel gradients of acetonitrile in buffered mobile phase. A new approach was proposed to optimize matching segmented gradient profiles in the two dimensions. An algorithm was developed for automatic corrections of the shifts in retention in the second dimension induced by the parallel two-dimensional gradient operation technique. Using the porous-shell C18 column in the second dimension at elevated temperature (60 degrees C) and high pressure (480 bar) with optimized segmented profiles of the parallel gradients in the two dimensions, the overall separation time for comprehensive LC x LC was reduced to 30 min.     

Characterization of impurities in sisomicin and netilmicin by liquid chromatography/mass spectrometry

The characterization of unknown impurities present in netilmicin and sisomicin by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. The volatile ion-pairing agent trifluoroacetic acid (TFA) was used for the retention of the main compounds and their impurities on a reversed-phase (RP) C18 column, because they are highly hydrophilic and basic compounds. The method showed good separation between netilmicin and its four potential related substances prescribed in the European Pharmacopoeia, which were identified by comparison of their retention times with those of the reference substances. Furthermore, in total 16 unknown impurities in a netilmicin sample and six in a sisomicin sample with unknown identity were detected. The structures of the unknown compounds were deduced based on comparison of fragmentation patterns with those of the reference substances investigated in LC/MSn experiments by the use of electrospray ion trap mass spectrometry.     

Development of an online capillary comprehensive 2D-LC system for the analysis of proteome samples

In the present contribution, a fully automated capillary comprehensive two-dimensional LC (LC×LC) method, for proteomic analysis, was developed for the first time. The investigated platform was characterized by the coupling of high-pH RP with low-pH RP separations thus ensuring the generation of high peak capacity despite the employment of identical stationary phases. The use of capillary columns in both dimensions allowed to reduce mobile-phase consumption and enhance sensitivity. Fraction transfer from the first to the second dimension was performed by means of two 2-position 6-port nano-switching valves, under stop-flow conditions. Values as high as 1208 and 955 were obtained for the theoretical and practical peak capacity, respectively. The investigated LC×LC system showed good retention time repeatibility with RSD values ranging from 0.8 to 6.0% for the first dimension and from 1.0 to 3.0% for the second dimension, respectively. RSD peak area values below 9.5% were also attained, thus demonstrating the precision of the LC×LC method employed.     

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.     

Stationary phase-based two-dimensional chromatography combining both covalent and noncovalent interactions on a single HPLC column

A new type of 2-D separation material was synthesized and studied. The material is suitable for 2-D chromatography utilizing both covalent and noncovalent interactions. The first dimension is boronate affinity chromatography, and the second dimension is RP chromatography (or vice versa). The polymeric media were prepared using p-vinylphenylboronic acid as the functional monomer. This monomer was selected due to the presence of the boronic acid group for the cis-diol/boronate interaction in boronate chromatography. Two crosslinkers were evaluated, namely ethylene glycol dimethacrylate and divinylbenzene. The crosslinker content was varied to maximize the polymer strength and the RP performance of the packed column. Several parameters were evaluated to define the optimum for polymer strength and column performance including crosslinker, porogen, initiator, and column-packing parameters. The polymer-based HPLC columns were successful in separating phenol, catechol, dimethylphthalate, and hydroquinone under RP conditions, and thus can be used as an RP HPLC column. The columns were also successful in separating catechol and adenosine under boronate chromatography conditions, and thus can be used as a boronate affinity column. Moreover, the two types of chromatography can be performed consecutively on the same column during one complete chromatographic run, making it a 2-D chromatography. Under these 2-D conditions, the catechol was separated from a mixture of phenol, catechol, dimethylphthalate, and hydroquinone; the adenosine ribonucleoside was separated from a mixture of adenosine ribonucleoside, adenosine deoxyribonucleoside, and uridine deoxyribonucleoside. This type of single-column 2-D HPLC eliminates the requirement of a complex and expensive multidimensional HPLC instrument and provides increased peak capacity for separation.     

A method of calculating the second dimension retention index in comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

A method was developed to calculate the second dimension retention index of comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC/TOF-MS) data using n-alkanes as reference compounds. The retention times of the C(7)-C(31) alkanes acquired during 24 isothermal experiments cover the 0-6s retention time area in the second dimension retention time space, which makes it possible to calculate the retention indices of target compounds from the corresponding retention time values without the extension of the retention space of the reference compounds. An empirical function was proposed to show the relationship among the second dimension retention time, the temperature of the second dimension column, and the carbon number of the n-alkanes. The proposed function is able to extend the second dimension retention time beyond the reference n-alkanes by increasing the carbon number. The extension of carbon numbers in reference n-alkanes up to two more carbon atoms introduces <10 retention index units (iu) of deviation. The effectiveness of using the proposed method was demonstrated by analyzing a mixture of compound standards in temperature programmed experiments using 6 different initial column temperatures. The standard deviation of the calculated retention index values of the compound standards fluctuated from 1 to 12 iu with a mean standard deviation of 5 iu.     

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.     

Direction of temperature gradient for normal-phase temperature gradient interaction chromatography in polystyrene fractionation

Temperature gradient interaction chromatography (TGIC) is a powerful technique for molecular weight fractionation of polymers, in which the interaction strength is controlled by varying the column temperature. In the present paper, the effects of the sign of the temperature dependence of the retention and the direction of the temperature gradient (raising or lowering) on TGIC in the normal-phase mode were studied for the molecular weight fractionation of polystyrene samples in organic mobile phases. It was found that a positive temperature gradient was effective in the system consisting of amino-modified silica (NH(2)) column and the eluent mixture of tetrahydrofuran and n-hexane where retention decreased with increasing temperature. A negative temperature gradient was effective for the systems consisting of a bare-silica column//chloroform/n-hexane and NH(2)-column//chloroform/n-hexane, where retention increased with increasing temperature. Increasing retention with increasing temperature has been found, so far, only for a water-soluble polymer (PEO) in an aqueous mobile phase in RP-TGIC.     

Effects of first dimension eluent composition in two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC×LC) has received a great deal of attention during the past few years because of its extraordinary resolving power. The biggest advantage of this technique is that very high peak capacities can be generated in a relatively short time. Numerous approaches to maximize the peak capacity in LC×LC have been employed. In this work we investigate the impact of the first dimension mobile phase on selectivity. LC×LC has several potential advantages over one-dimensional LC (1DLC) in that unconventional solvents, at least in reversed-phase LC, can be used. For example, solvents which strongly adsorb in the UV in the first dimension are not problematic in LC×LC. This so because the UV detector is placed after the second dimensional column, as pulses of the first dimension eluent arrive at the second dimensional column, they elute well before the solutes of interest and therefore do not interfere at all with detection of solute peaks. So far, the most widely used solvents in reversed-phase 1DLC are methanol and acetonitrile. However, the "UV advantage" of 2DLC allows us to employ UV active solvents, such as acetone. We compare their differential selectivities to that of acetonitrile for the separation of 23 indole acetic acids of interest in plant biology. We also apply them to the separation of a maize seed extract, a very complex sample. In both sample sets, mobile phase composition can be an important parameter to increase the orthogonality of the two dimensions and thus, to increase the effective peak capacity of LC×LC.     

Screening active anti‐breast cancer compounds from Cortex Magnolia officinalis by 2D LC–MS

Most of the anti‐breast cancer drugs are often limited owing to drug resistance and serious adverse reactions. Therefore, development of more targeted and low toxic drugs from traditional Chinese medicines for breast cancer are needed. At the same time, establishment of fast and effective drug screening methods are urgently required. We describe here a 2D LC method of MDA‐MB‐231 cell membrane chromatography combined with HPLC/MS for recognition, separation, and identification of target components from traditional Chinese medicine Cortex Magnolia officinalis. The MDA‐MB‐231 cells membrane was used to prepare the chromatographic stationary phase in the first dimension. The active compounds had a retention characteristic on the cell membrane chromatography model (10 × 2.0 mm, 5 μm). The retention fractions were enriched using an online C₁₈ column (10 × 1.0 mm, 5 μm) and were analyzed by the second dimension RP chromatography. Finally, the activity of the retention fractions was tested through in vitro experiments. Results showed that the retention fractions were honokiol and magnolol and the inhibition rate on MDA‐MB‐231 cell growth were 23 and 64 μM, respectively. These results support the conclusion that this coupled analytical technique could be an efficient method in drug discovery.     

利用后凝胶化反应制备低吸附性反相填料

在硅胶基质的反相填料 (如 C18、C8、C4 等 )上 ,蛋白质的变性 ,碱性分子峰展宽、拖尾甚至完全吸附等常被归因为硅羟基产生的次级效应 (Secondary Effect) [1～ 3] .我们认为 ,除填料的孔径效应外 [4 ] ,杂质离子与残余硅羟基的协同效应是导致此类问题的直接原因 ,如金属离子直接作用于溶质以及金属离子和硅羟基的远程作用力导致的溶质变性等 .为解决这一问题 ,可采用高反应性的新硅烷化试剂 ,以尽可能将硅羟基完全屏蔽 [5] ,或以封端试剂消除残余的硅羟基 .但是 ,用硅烷化试剂封端 (End-capping)在多数情况下并不能完全覆盖表面硅羟基 ,…