Separation of seventeen 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and dibenzofurans and 12 dioxin-like polychlorinated biphenyls by comprehensive two-dimensional gas chromatography with electron-capture detection

Comprehensive two-dimensional gas chromatography (GC x GC) with electron-capture detection (ECD) has been optimized for the separation of seventeen 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and dibenzofurans and 12 dioxin-like polychlorinated biphenyls, with emphasis on the selection of the first- and second-dimension, commercially available, columns. When eight second-dimension columns were subsequently combined with a 100% methylpolysiloxane stationary phase (DB-1) in the first dimension to create orthogonal conditions, a complete separation of all congeners with different TEF values was obtained with two column combinations, DB-1 x VF-23 and DB-1 x LC-50. When other types of first-dimension columns were used (and orthogonality was partly sacrificed), a DB-XLB column combined with 007-65HT, VF-23 and LC-50 was found to provide a complete separation of all 29 priority congeners. Next, the potential of these three column combinations for real-life analysis was preliminarily studied. With a spiked and fractionated milk extract, DB-XLB x LC-50 was found to be the most powerful column combination, because of the good separation of the 29 priority congeners from each other as well as from the matrix constituents. Quantitative performance (close to three-order linearity; LODs, 30-150 fg injected; R.S.D.s, 1.5-6.5% (n = 10)) was satisfactory.     

Overloading of the second-dimension column in comprehensive two-dimensional gas chromatography

Comprehensive two-dimensional gas chromatography (GC x GC) is based on a coupling of two GC columns of different characteristics by means of a device that allows portions of the effluent from the primary column to be injected onto the second dimension column for an additional separation. The time available for the separation in the second-dimension column is very short. Thus, this separation should be very efficient. The vast majority of GC x GC practitioners use very narrow bore columns for the second dimension. While this approach is justified in principle, if peaks in the second dimension overload this column, its peak capacity is severely reduced. A series of second-dimension columns of varying internal diameters, but similar phase ratios, were used to study these effects. The results indicate that 250 microm columns often provide comparable second dimension peak widths to 100 microm columns, while at the same time being less prone to overloading, indicating that they may often be a better choice than smaller diameter columns in the second dimension of GC x GC systems.     

Comprehensive two-dimensional liquid chromatography with parallel gradients for separation of phenolic and flavone antioxidants

Various combinations of PEG-silica, phenyl-silica and C18 columns in a single-column or serial (tandem) arrangement in the first dimension and a monolithic Chromolith column in the second dimension were tested for comprehensive two-dimensional (2D) LCxLC separation of phenolic and flavone natural antioxidants. The combinations of different stationary phase chemistries provided low selectivity correlations between the first-dimension and the second-dimension separation systems. Improvement in system orthogonality, bandwidths suppression, more regular band distribution over the whole 2D retention plane and increased peak capacity in different 2D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2D separation time range. Instead of two sampling loops, two alternating trapping XTerra columns were used for sample fraction transfer from the first-dimension column to the second dimension. Stronger retention on the XTerra columns in comparison to PEG-silica or phenyl-silica columns in the first dimension allowed using focusing of sample fractions in narrow zones on the top of a trapping column and back-flushing into the second dimension in a very low volume of the mobile phase. This fraction transfer modulation provided significant bandwidth suppression in the second dimension. 2D systems with optimized stationary phase selectivity, parallel gradients and fraction transfer modulation using two trapping columns were applied for the analysis of natural antioxidants in beer and wine samples.     

Effect of first-dimension column film thickness on comprehensive two-dimensional gas chromatographic separation

In comprehensive two-dimensional gas chromatography (GC x GC), samples experience two-dimensional separation implemented by a modulator which helps preserve the first-dimension separation and facilitates the second-dimension separation by periodically collecting, focusing and launching the material from the primary column onto the secondary column with a different stationary phase. Column overloading in GC x GC is a considerable problem, aggravated by the fact that two columns are involved. Broad first-dimension peaks of an analyte help produce smaller fractions of the analyte in the second-dimension, reducing the chance of secondary column overloading. One of the means to generate broad peaks in the first-dimension is to use thick film primary columns. A series of primary columns of various film thickness were tested in the study, and the results indicate that when other conditions are kept constant, 1 microm film columns often provide better resolution in both first and second-dimension but at the expense of a much longer separation time; 0.1 microm is clearly inadequate for GC x GC separation; 0.5 and 0.25 microm film columns seem to be the best compromises.     

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。     

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.     

Solid phase microextraction-comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry for the analysis of honey volatiles

Head-space solid phase microextration (SPME), followed by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-TOFMS), has been implemented for the analysis of honey volatiles, with emphasis on the optimal selection of SPME fibre and the first- and second-dimension GC capillaries. From seven SPME fibres investigated, a divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 microm fibre provided the best sorption capacity and the broadest range of volatiles extracted from the headspace of a mixed honey sample. A combination of DB-5ms x SUPELCOWAX 10 columns enabled the best resolution of sample components compared to the other two tested column configurations. Employing this powerful analytical strategy led to the identification of 164 volatile compounds present in a honey mixture during a 19-min GC run. Combination of this simple and inexpensive SPME-based sampling/concentration technique with the advanced separation/identification approach represented by GCxGC-TOFMS allows a rapid and comprehensive examination of the honey volatiles profile. In this way, the laboratory sample throughput can be increased significantly and, at the same time, the risk of erroneous identification, which cannot be avoided in one-dimensional GC separation, is minimised.     

Evaluation of the separation characteristics of application-specific (volatile organic compounds) open-tubular columns for gas chromatography

The solvation parameter model is used to characterize the separation characteristics of two application-specific open-tubular columns (Rtx-Volatiles and Rtx-VGC) and a general purpose column for the separation of volatile organic compounds (DB-WAXetr) at five equally spaced temperatures over the range 60-140 degrees C. System constant differences and retention factor correlation plots are then used to determine selectivity differences between the above columns and their closest neighbors in a large database of system constants and retention factors for forty-four open-tubular columns. The Rtx-Volatiles column is shown to have separation characteristics predicted for a poly(dimethyldiphenylsiloxane) stationary phase containing about 16% diphenylsiloxane monomer. The Rtx-VGC column has separation properties similar to the poly(cyanopropylphenyldimethylsiloxane) stationary phase containing 14% cyanopropylphenylsiloxane monomer DB-1701 for non-polar and dipolar/polarizable compounds but significantly different characteristics for the separation of hydrogen-bond acids. For all practical purposes the DB-WAXetr column is shown to be selectivity equivalent to poly(ethylene glycol) columns prepared using different chemistries for bonding and immobilizing the stationary phase. Principal component analysis and cluster analysis are then used to classify the system constants for the above columns and a sub-database of eleven open-tubular columns (DB-1, HP-5, DB-VRX, Rtx-20, DB-35, Rtx-50, Rtx-65, DB-1301, DB-1701, DB-200, and DB-624) commonly used for the separation of volatile organic compounds. A rationale basis for column selection based on differences in intermolecular interactions is presented as an aid to method development for the separation of volatile organic compounds.     

Development and optimization of a system for comprehensive two-dimensional liquid chromatography with UV and mass spectrometric detection for the separation of complex samples by multi-step gradient elution

Comprehensive two-dimensional liquid chromatography (LC x LC) is a powerful tool for the separation of complex biological samples. This technique offers the advantage of simplified automation and greater reproducibility in a shorter analysis time than off-line two-dimensional separation systems. In the present study, an LC x LC system is developed enabling simultaneous UV and MS detection, and which can be easily converted to a conventional reversed-phase LC-UV/MS system. In LC x LC, a 60-min reversed-phase LC separation with a linear solvent gradient in the first dimension is coupled to a second-dimension separation on a mixed-mode cation-exchange/reversed-phase column with a modulation time of 60s. The isocratic separation in the second-dimension column is optimized by the use of a multi-step gradient where the organic and the ionic modifier are varied independently. Intraday (n=3) and interday (n=4) variability of the retention times were evaluated with the complete system and found to be 0.5% and 0.7%, respectively. Good linearity was observed in calibration curves for three different compounds varying in polarity.     

Comparing columns for gas chromatography with the two-parameter model for retention prediction

The retention times of selected compounds in temperature programmed gas chromatography were predicted using a two-parameter model, on the basis of thermodynamic data obtained from isothermal runs on seven capillary columns, primarily substituted with 5% diphenylsiloxane. The scope for using thermodynamic data obtained from isothermal runs on one column to optimize separation on a different column or a different instrument setup was investigated. Additionally, the predictive utility of thermodynamic data obtained using a DB-5 column that had been in use for three years was compared to that of a new column of the same model. It was found that satisfactory separation could be achieved on one capillary column or instrument setup on the basis of thermodynamic data obtained using a different column or instrument set-up.     

Optimization of separation in two-dimensional high-performance liquid chromatography by adjusting phase system selectivity and using programmed elution techniques

The overall peak capacity in comprehensive two-dimensional liquid chromatographic (LC x LC) separation can be considerably increased using efficient columns and carefully optimized mobile phases providing large differences in the retention mechanisms and separation selectivity between the first and the second dimension. Gradient-elution operation and fraction-transfer modulation by matching the retention and the elution strength of the mobile phases in the two dimensions are useful means to suppress the band broadening in the second dimension and to increase the number of sample compounds separated in LC x LC. Matching parallel gradients in the first and second dimension eliminate the necessity of second-dimension column re-equilibration after the independent gradient runs for each fraction, increase the use of the available second-dimension separation time and can significantly improve the regularity of the coverage of the available retention space in LC x LC separations, especially with the first- and second-dimension systems showing partial selectivity correlations. Systematic development of an LC x LC method with parallel two-dimensional gradients was applied for separation of phenolic acids and flavone compounds. Several types of bonded C18, amide, phenyl, pentafluorophenyl and poly(ethylene glycol) columns were compared using the linear free energy relationship method to find suitable column combination with low correlation of retention of representative standards. The phase systems were optimized step-by-step to find the mobile phases and gradients providing best separation selectivity for phenolic compounds. The optimization of simultaneous parallel gradients in the first and second dimension resulted in significant improvement in the utilization of the available two-dimensional retention space.     

Simple 2D-HPLC using a monolithic silica column for peptide separation

Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.     

Short capillary column for the separation and identification of mononitrofluoranthene and mononitropyrene isomers present in air and emission samples

A 15 m DB-17 fused-silica capillary column has been used to achieve the separation of all mononitrofluorarthene and mononitropyrene isomers. Linear Retention Indices and Relative Retention times for each component have been measured and the values were compared with those observed on DB-5 columns, commonly used for this separation. Increased polarity of the liquid phase results in better separation of some key isomeric pairs (such as 2-, 3-nitrofluoranthene and 1-nitropyrene, 8-nitrofluoranthene), difficult to resolve on DB-5 columns. Practical applications are reported. They include the analysis of extracts from emission samples (diesel exhaust and particles emitted by a factory manufacturing carbon electrodes) as well as atmospheric dust sampled in a urban area under different conditions.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

Theoretical advantages and drawbacks of on-line,multidimensional liquid chromatography using multiple columns operated in parallel

The theoretical advantages and drawbacks of using a multiple-, parallel column approach in on-line multidimensional liquid chromatography systems were investigated. Much time or peak capacity can be gained with the use of multiple parallel columns at the second-dimension while the aggregate time of separation increases only by the increment of the gradient time of the second-dimension. Multidimensional chromatographic systems are now used to perform many tasks ranging from routine, fast analyses to specialized, arduous separations. In this work, we focus on the advantages of a multiple, parallel columns approach to on-line multidimensional liquid chromatography systems. Calculations of the achievable peak capacities were made as functions of the number of columns operated in parallel. Increasing the number of second-dimension columns from one to two or three causes the largest increase in peak capacity with only a slight increase of aggregate time. We also present some practical aspects to consider when attempting multidimensional separations with multiple columns operated in parallel.     

Flame ionization detection after splitting the water effluent in subcritical water chromatography

The coupling of subcritical water separation with flame ionization detection (FID) in the split mode has been investigated in this study. In order to keep the FID system stable during subcritical water separation, a Tee union was connected between the separation column and the FID system to split the water flow. The ratio of the water flow to the FID system over the flow-rate to a waste bottle varied depending on the dimension of capillary tubings and the total water flow-rate used. Separations of several carbohydrates, carboxylic acids, and amino acids were performed on commercially available columns using a laboratory-made subcritical water chromatography-FID system. The FID system was very stable in this split mode even at total flow-rate as high as 1.24 ml/min. The linear dynamic range was up to three orders of magnitude and the limit of detection (LOD) ranged from 38 to 111 ng (306-925 ng/microl injected) with split ratios of approximately 1:10 to approximately 1:17 (FID/waste bottle) for several analytes studied. However, the LOD can be significantly lowered by adjusting the dimensions of the restrictors to allow a higher percentage of the total flow to the FID system.     

Columns and optimum gradient conditions for fast second-dimension separations in comprehensive two-dimensional liquid chromatography

Gradient elution provides significantly higher peak capacity in comparison to the isocratic elution mode, hence it is very useful in online comprehensive two-dimensional liquid chromatography (LC). We compared suitability of five commercial core-shell columns and one monolithic column for fast gradients in the second LC dimension, where the time of separation is strictly limited by the fraction cycle time. In two-dimensional reversed-phase systems with partially correlated retention, the resolution, the peak capacity, and the regularity of coverage of the second-dimension retention space can be improved by appropriate adjusting the gradient time and the gradient range to suit the sample properties. We developed a new strategy for adjusting the gradient mobile phase composition range in the second-dimension, employing the retention data of representative sample standards characterizing the sample properties, which can be calibrated using the reference alkylbenzene series. Optimized second-dimension gradients with single-step or segmented profiles covering two or more fraction ranges, employed for the separation of subsequent fractions from the first-dimension, improve significantly the resolution, the separation time, and the regularity of coverage of the two-dimensional retention plane. The approach was applied to the two-dimensional comprehensive separation of phenolic acids and flavonoid compounds occurring as natural antioxidants.     

Evaluation of fused-silica capillary columns for GC/ECD analysis of chlorinated hydrocarbons listed in EPA method 8120

Four mega-bore, one wide-bore, and one narrow-bore fused-silica capillary columns were evaluated for their applicability to the GC/ECD analysis of 22 chlorinated hydrocarbons, some of which are currently targeted by EPA Method 8120. No one column can resolve all 22 compounds investigated here. Four compounds (two pairs) are coeluting on the SPB-35, DB-210, DB-WAX, and DB-519 fused-silica capillary columns, five compounds (two groups) are coeluting on the DB-1301 fused-silica capillary column, and ten compounds (five pairs) are coeluting on the SPB-5 fused-silica capillary column. The analysis time varies between 30 and 50 min. The order of elution of the chlorinated benzenes seems to depend on their boiling points rather than on the polarity of the liquid phase. The retention times of an additional nine chlorinated toluenes, eight chlorinated xylenes, and five chlorinated naphthalenes are also reported. Electron capture detector linearity is reported for the DB-210 fused-silica capillary column. Five brominated compounds were investigated as possible internal standards for Method 8120.     

On-Line multitasking analytical proteomics: how to separate, reduce, alkylate and digest whole proteins in an on-Line multidimensional chromatography system coupled to MS

An on-Line multidimensional system has been developed, consisting of pH gradient strong anion exchange chromatography of native proteins in the first dimension with subsequent trapping and on-column reduction/alkylation on C4 trap columns and RP separation of the alkylated proteins in the second dimension followed by on-column tryptic digestion and electrospray MS detection. The system was evaluated using model proteins and a human urine sample. Compared to the commonly used in-solution alkylation method, the developed on-column method provides an equivalent efficiency. The recovery from the C4 trap columns of the alkylated proteins relative to the native state was from 94 to 102%. On-column tryptic digestion was satisfactory for many, but not for all proteins. The whole analytical procedure was performed on-Line with packed capillary columns for a total time of 320 min for the first ion exchange fraction, with additional 60 min for each subsequent fraction.     

Stroboscopic sampling in comprehensive high-performance liquid chromatography-capillary electrophoresis via a pneumatic sampler

A new approach is presented to solve the problem of a long separation time in the second dimension of comprehensive two-dimensional chromatography. The need for a rapid separation in the second column is overcome by repeating analysis of a sample many times. In each of these individual analysis cases the sample is injected into the first dimension column and after a delay a low amount of the effluent at the end of the first column is sampled to the second-dimensional column. The time interval between the samplings from the first column to the second column is constantly increased. Thus, the system enables a comprehensive analysis of the effluent emerging from the first into the second column. This approach, which we call stroboscopic sampling, is tested for coupling high-performance liquid chromatography (HPLC) to capillary electrophoresis (CE) by an interface which operates on the principle of transporting the effluent from the HPLC column to the capillary inlet by small pressure pulses (0.5 MPa). The performance of the interface for accomplishing the comprehensive HPLC-CE analysis was demonstrated for an on-line connection of a short ion-exchange column and an ion-exclusion column to the CE capillary.