Two-dimensional supercritical fluid chromatography/mass spectrometry for the enantiomeric analysis and purification of pharmaceutical samples

A new analytical two-dimensional supercritical fluid chromatography/mass spectrometry system (2D SFC/SFC/MS) has been designed and implemented to enhance the efficiency and quality of analytical support in drug discovery. The system consists of a Berger analytical SFC pump and a modifier pump, a Waters ZQ 2000 mass spectrometer, a set of switching valves, and a custom software program. The system integrates achiral and chiral separations into a single run to perform enantiomeric analysis and separation of a racemic compound from a complex mixture without prior clean up. The achiral chromatography in the first dimension separates the racemate from all other impurities, such as un-reacted starting materials and by-products. Mass-triggered fractionation is used to selectively fractionate the targeted racemic compound based on its molecular weight. The purified racemate from the achiral chromatography in the first dimension is then transferred to the chiral column in the second dimension to conduct the enantiomeric separation and analysis. A control software program, we coined SFC2D, was developed and integrated with MassLynx to retrieve acquisition status, current sample information, and real time mass spectrometric data as they are acquired. The SFC2D program also monitors the target ion signal to carry out mass-triggered fractionation by switching the valve to fractionate the desired peak. The 2D SFC/SFC/MS system uses one CO(2) pump and one modifier pump for both first and second dimension chromatographic separations using either gradient or isocratic elution. Similarly, a preparative 2D SFC/SFC/MS system has been constructed by modifying an existing Waters preparative LC/MS system. All components except the back pressure regulator are from the original LC/MS system. Applications of the 2D SFC/SFC/MS methods to the separation and the analysis of racemic pharmaceutical samples in complex mixtures demonstrated that an achiral separation (in first dimension) and a chiral separation (in second dimension) can be successfully combined into a single, streamlined process both in analytical and preparative scale.     

Retention on non-polar adsorbents in liquid—solid chromatography Effect of grafted alkyl chains

The retention of 39 molecular probes was measured on chemically bonded dense layers of (3,3-dimethylbutyl)dimethylsiloxy (DMB) and tetradecyldimethylsiloxy (C₁₄) substituents on silica as a function of the composition of the binary acetonitrile-water eluent. The sign of the “associated system peak” was also noted. The composition dependence of retention, measured as areal retention volume, could be described on both non-polar stationary phases by the two-parameter Snyder-Soczewinski equation and by the three parameter Schoenmakers equation in a broad but restricted composition range. The areal retention volume on the surface with grafted alkyl chains was equal to or higher than that on the non-swellable, smooth, non-polar DMB surface. Additional retention on the C₁₄ surface increased with increasing adsorption force (retention) on the DMB surface and it was a function of the composition of the eluent. A possible interpretation of the sign of the “associated system peak” generated by the injection of a pure solute is also given in terms of salvation of the solute in the mobile phase and in terms of the modification of the non-polar adsorbent by the adsorbed solute.     

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.     

Nano-flow multidimensional liquid chromatography with electrospray ionization time-of-flight mass spectrometry for proteome analysis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the top five cancers with the highest incident of a disease worldwide. To understand the mechanisms of hepatocarcinogenesis, proteomics analysis provides a powerful tool to identify proteins that associate with HCC. We developed a two-step procedure for mapping of HCC proteomics. In the first step, in order to simplify the complexity of proteomics of HCC, the subfractionation of complex protein mixtures in HCC into “subproteomes” is presented based on the solubility of protein. While in the second step an automate comprehensive two-dimensional (2D) separation system, coupling strong cation-exchange (SCX) in the first dimension with capillary reversed-phase chromatography (cRPLC) in the second dimension is developed further to separate and analyze proteins associated with HCC. By using this system, complex sample can be injected, desalted, separated and analyzed in complete automatization. The procedure for proteomics analysis was found to be applied for proteins with great molecular mass (>100 000), small molecular mass (<20 000), highly basic (pI > 9.5) and hydrophobicity, which are not well resolved in 2D-gel electrophoresis. In total 229 proteins were identified by using the described proteomics platform. Among them, several proteins related to the process of carcinogenesis were investigated further.     

Concerning the theory of chirality functions : Part I. Construction and discussion of chirality functions by means of [2.2] metacyclophanes

By means of [2.2]metacyclophane (skeleton symmetry C_2h) the construction of qualitatively-complete chirality functions via the first and second “Naherungsansatze” is demonstrated. In this context, a detailed comment concerning the algebraic theory of chirality functions is given. As the first term of the chirality function (“abridged Ansatz”), both methods furnish a simple superposition rule (“quadrant rule”). The second component as deduced via the method of polynomials is of third degree in ligand specific parameters. A particular property of the second approximation method is derived. The consequences with respect to observables of the chirality phenomenon, in the case of the approximations being valid, are discussed.     

Preparative separation of anti‐oxidative constituents from Rubia cordifolia by column‐switching counter‐current chromatography

An effective column‐switching counter‐current chromatography (CCC) protocol combining stepwise elution mode was successfully developed for simultaneous and preparative separation of anti‐oxidative components from ethyl acetate extract of traditional Chinese herbal medicine Rubia cordifolia. The column‐switching CCC system was interfaced by a commercial low‐pressure six‐port switching valve equipped with a sample loop, allowing large volume introduction from the first dimension (1^st‐D) to the second dimension (2^nd‐D). Moreover, to extend the polarity window, three biphasic liquid systems composed of n‐hexane/ethyl acetate/methanol/water (1:2:1:2, 2:3:2:3, 5:6:5:6 v/v) were employed using stepwise elution mode in the 1^st‐D. By valve switching technique the whole interested region of 1^st‐D could be introduced to second dimension for further separation with the solvent system 5:5:4:6 v/v. Using the present column‐switching CCC protocol, 500 mg of crude R. cordifolia extract were separated, producing milligram‐amounts of four anti‐oxidative components over 90% pure. Structures of purified compounds were identified by ¹H and ¹³C NMR.     

Comprehensive two-dimensional liquid chromatography applying two parallel columns in the second dimension

The design of a new interface for comprehensive two-dimensional liquid chromatography (LC x LC) is described. To the conventionally used LC x LC system with the loop-type interface consisting of a two-position/ten-port switching valve equipped with two loops, an extra two-position/ten-port switching valve, a detector, a pump and a second column placed in parallel with the column in the second dimension, are added. The features of the interface are that the separation space in the second dimension is significantly enlarged and that the number of fractions transferred from the first to the second dimension can be increased, reducing the risk to lose resolution of the primary dimension. The potential of the system in NPLC x 2RPLC is illustrated with the analysis of a standard mixture and a lemon oil extract. For the lemon oil analysis, the effective peak capacity was increased from 437 using a conventional interface to 1095 with the new interface. RPLC x 2RPLC in combination with reduced modulation times was applied to the analysis of steroids and to the detection of impurities at the 0.05% relative concentration level in a sulfonamide drug sample.     

Multidimensional liquid chromatography system with an innovative solvent evaporation interface

An orthogonal two-dimensional liquid chromatographic (2D-LC) system was developed by using a vacuum-evaporation loop-type valve interface. Normal-phase liquid chromatography (NPLC) with a bonded CN phase column was used as the first dimension, and reversed-phase liquid chromatography (RPLC) with a C(18) column was used as the second dimension. All the solvents in the loop of the interface were evaporated at 90 degrees C under vacuum conditions, leaving the analytes on the inner wall of the loop. The mobile phase of the second dimension dissolved the analytes in the loop and injected them onto the secondary column, allowing an on-line solvent exchange of a selected fraction from the first dimension to the second dimension. The chromatographic resolution of analytes on the two dimensions was maintained at their optimal condition. Sample loss due to evaporation in the interface was observed that depended on the boiling point of the compound. Separation of sixteen polycyclic aromatic hydrocarbon mixtures and a traditional Chinese medicine Angelica dahurica was demonstrated.     

Serial displacement chromatofocusing and its applications in multidimensional chromatography and gel electrophoresis: II. Experimental results

Part I of this study investigated the theory and basic characteristics of “serial displacement chromatofocusing” (SDC). In Part II of this study, SDC is applied to two prototype applications which have potential uses in proteomics and related areas involving the analysis of complex analyte mixtures. In the first application, SDC was used as a prefractionation method prior to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate a human prostate cancer cell lysate. It was observed that the resolution achieved in narrow-pI-range 2D-PAGE was improved when using SDC prefractionation, so that SDC may be useful as a low-cost, high-speed, and highly scalable alternative to electrophoretic prefractionation methods for 2D-PAGE. The second application involves the use of SDC as the first dimension, and reversed-phase chromatography as the second dimension, to produce a novel, fully automated, two-dimensional high-performance liquid chromatography technique. The method was shown to have performance advantages over one-dimensional reversed-phase chromatography for peptide separations.     

Protein mapping by two-dimensional high performance liquid chromatography

Current developments in drug discovery in the pharmaceutical industry require highly efficient analytical systems for protein mapping providing high resolution, robustness, sensitivity, reproducibility and a high throughput of samples. The potential of two-dimensional (2D) HPLC as a complementary method to 2D-gel electrophoresis is investigated, especially in view of speed and repeatability. The method will be applied for proteins of a molecular mass <20 000 which are not well resolved in 2D-gel electrophoresis. The 2D-HPLC system described in this work consisted of anion- or cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. We used a comprehensive two-dimensional approach based on different separation speeds. In the first dimension 2.5 microm polymeric beads bonded with diethylaminoethyl and sulfonic acid groups, respectively, were applied as ion exchangers and operated at a flow-rate of 1 ml/min. To achieve very high-speed and high-resolution separations in the second dimension, short columns of 14 x 4.6 mm I.D. with 1.5 microm n-octadecyl bonded, non-porous silica packings were chosen and operated at a flow-rate of 2.5 ml/min. Two reversed-phase columns were used in parallel in the second dimension. The analyte fractions from the ion-exchange column were transferred alternatively to one of the two reversed-phase columns using a 10-port switching valve. The analytes were deposited in an on-column focusing mode on top of one column while the analytes on the second column were eluted. Proteins, which were not completely resolved in the first dimension can, in most cases, be baseline-separated in the second dimension. The total value of peak capacity was calculated to 600. Fully unattended overnight runs for repeatability studies proved the applicability of the system. The values for the relative standard deviation (RSD) of the retention times of proteins were less than 1% (n = 15), while the RSDs of the peak areas were less than 15% (n = 15) on average. The limit of detection was 300 ng of protein on average and decreased to 50 ng for ovalbumin. The 2D-HPLC system offered high-resolution protein separations with a total analysis time of less than 20 min, equivalent to the run time of the first dimension.     

Interface‐free two‐dimensional heart‐cutting capillary electrophoresis for the separation and stacking of anionic and neutral analytes

Interface‐free two‐dimensional heart‐cutting capillary electrophoresis for two different classes of analytes (anionic and neutral) in a single capillary is presented. Simultaneous sample stacking and orthogonal separation were demonstrated. The anionic species were first analyzed by capillary zone electrophoresis in the first dimension. Then, the neutral compounds were separated in the second dimension by micellar electrokinetic chromatography using the common anionic surfactant sodium dodecyl sulfate. The first and second dimensions occurred automatically without changing the electrolyte and without polarity switching. Artificial mixtures (five anions and four neutral compounds) were successfully analyzed with sensitivity enhancement factors from 7 to 28. The orthogonal separation was complete within 8 min. Some analytical features and application to a spiked real river water sample were also studied.     

Multidimensional gas chromatography using a double cool-strand interface

Commercial interfaces for multidimensional gas chromatography (MDGC) are based either on a valve or a pneumatic switching between columns. Both exhibit significant drawbacks and only few suppliers exist. An extremely simple interface has been set up to overcome these limitations without requiring any pneumatic control or valves switching. This new MDGC design is based on the cryo-control of the analyte transfer from the first to the second column through two cool strands of a capillary. This technique is simple to implement and does not require any special column connections. Applications involve non-polar/polar phase combinations, as well as chiral analysis, hyphenation to a conventional mass spectrometer, and olfactometric detection. In contrast to conventional MDGC configuration, the present configuration allows the use of a single oven to operate both columns at different temperatures.     

Using perdeuterated surfactant micelles to resolve mixture components in diffusion‐ordered NMR spectroscopy

Diffusion‐ordered NMR spectroscopy resolves mixture components on the basis of differences in their respective diffusion coefficients or molecular sizes. However, when components have near‐identical diffusion coefficients, they are not resolved in the diffusion dimension of a diffusion‐ordered spectroscopy (DOSY) spectrum. Adding surfactant micelles to these mixtures has been shown to enhance resolution when the component molecules interact differentially with the micelles. This approach is similar to that used in electrokinetic chromatography (EKC) where modifiers like micelles or polymers are used to enhance the separation of mixture components. In this study, perdeuterated surfactants are added to analyte mixtures studied with the DOSY technique. Since no micelle resonances appear in the mixture spectra, the difficulty associated with performing biexponential analyses in spectral regions where analyte and surfactant resonances overlap is avoided. The approach is demonstrated using mixtures of peptides with near‐identical diffusion coefficients. Copyright © 2008 John Wiley & Sons, Ltd.     

Combining monolithic and hollow capillary columns under conditions of two-dimensional gas chromatography

A monolithic column with the optimum structure of its monolithic layer is considered as a second dimension column under the conditions of two-dimensional gas chromatography. It is shown that despite the considerable difference between the working pressures of different columns, sample transfer from a hollow capillary column to a monolithic column is possible when a looped switching valve is used. It is found that the additional broadening of the chromatographic zone observed during sample transfer can be effectively suppressed by using an additional flow splitter. At the same time, reducing the volume of the switching valve loop is inefficient and does not allow effective separation by the monolithic column.     

On the reaction mechanism of gamma-ray solid-state copolymerization

The gamma-ray induced solid-state polymerization of binary mixtures consisting of a maleimide derivative as the first component and acenaphthylene or trans-stilbene as the second component was investigated regarding the occurrence of copolymerization. The binary solid mixture of these compounds exhibited a phase-equilibrium diagram including a simple eutectic mixture, and the irradiation caused the formation of a random copolymer together with that of homopolymers of component comonomers. The reaction rates and the composition of products suggested that the mobility of monomer molecules in the crystals affected the solid-state polymerization significantly. The solid-state copolymerization was supposed to take place by the diffusion of comonomer molecules via a vapor phase to the nuclei where the copolymerization proceeded.     

Liquid exclusion-adsorption chromatography,a new technique for isocratic separation of nonionic surfactants. IV. Two-dimensional separation of fatty alcohol ethoxylates with focusing of fractions

Fatty alcohol ethoxylates can be analyzed using a combination of liquid chromatography under critical conditions as the first dimension and liquid exclusion-adsorption chromatography as the second dimension. Transfer of fractions from the first to the second dimension is achieved using the full adsorption-desorption (FAD) technique. The peaks of interest in the first dimension are trapped on a short precolumn before injecting them into the second dimension. Full adsorption is achieved by increasing the water content in the mobile phase before the FAD column. When the fractions are desorbed by switching to the mobile phase of the second dimension, they are focused and reconcentrated. In this way, a full resolution of oligomers is achieved. As both dimensions are run in isocratic mode, density and refractive index detection can be applied, which allows an accurate quantitation.     

Simultaneous quantification of five proteins and seven additives in dairy products with a heart‐cutting two‐dimensional liquid chromatography method

A heart‐cutting two‐dimensional high‐performance liquid chromatography method was developed to simultaneously quantify five major proteins and seven food additives (maltol, ethyl maltol, vanillin, ethyl vanillin, benzoic acid, sorbic acid, and saccharin sodium) in milk and milk powders. In this two‐dimensional system, a Venusil XBP‐C4 column was selected in the first dimension for protein separation, and a Hypersil ODS‐2 C18 column was employed in the second dimension for additive separation; a two‐position, six‐port switching valve was used to transfer the targets (additives) from the first dimension to the second dimension. Method validation consisted of selectivity, response function, linearity, precision, sensitivity, and recovery. In addition, a conventional one‐dimensional high‐performance liquid chromatography method was also tested for comparison. The two‐dimensional method resulted in significantly improved recovery of the food additives compared to the conventional method (90.6–105.4% and 65.5–86.5%, respectively). Furthermore, this novel method has a simple one‐step sample preparation procedure, which shortens the analysis time, resulting in more efficient analysis and less solvent usage.     

Buffer gas additives (modifiers/shift reagents) in ion mobility spectrometry: Applications,predictions of mobility shifts,and influence of interaction energy and structure

Ion mobility spectrometry (IMS) is an analytical technique used for fast and sensitive detection of illegal substances in customs and airports, diagnosis of diseases through detection of metabolites in breath, fundamental studies in physics and chemistry, space exploration, and many more applications. Ion mobility spectrometry separates ions in the gas‐phase drifting under an electric field according to their size to charge ratio. Ion mobility spectrometry disadvantages are false positives that delay transportation, compromise patient's health and other negative issues when IMS is used for detection. To prevent false positives, IMS measures the ion mobilities in 2 different conditions, in pure buffer gas or when shift reagents (SRs) are introduced in this gas, providing 2 different characteristic properties of the ion and increasing the chances of right identification. Mobility shifts with the introduction of SRs in the buffer gas are due to clustering of analyte ions with SRs. Effective SRs are polar volatile compounds with free electron pairs with a tendency to form clusters with the analyte ion. Formation of clusters is favored by formation of stable analyte ion‐SR hydrogen bonds, high analytes' proton affinity, and low steric hindrance in the ion charge while stabilization of ion charge by resonance may disfavor it. Inductive effects and the number of adduction sites also affect cluster formation. The prediction of IMS separations of overlapping peaks is important because it simplifies a trial and error procedure. Doping experiments to simplify IMS spectra by changing the ion‐analyte reactions forming the so‐called alternative reactant ions are not considered in this review and techniques other than drift tube IMS are marginally covered.     

Comprehensive supercritical fluid chromatography × reversed phase liquid chromatography for the analysis of the fatty acids in fish oil

The separation of the phenacyl esters of the fatty acids originating from a fish oil extract by means of a comprehensive analysis using silver-ion (SI) supercritical fluid chromatography (SFC) and reversed phase liquid chromatography (RP-LC) in the first and second dimensions, respectively, is described. The combination ensured a high orthogonality and peak capacity, particularly when compared to the comprehensive RP-LC × 2RP-LC separation achieved by using a configuration with two columns in parallel in the second dimension. The construction of the SI-SFC × RP-LC interface consists of two two-position/ten-port switching valves, of which one is equipped with two loops packed with octadecyl silica (ODS) particles. Compared to the SFC × RP-LC configuration described in an earlier publication, the peak capacity in the second dimension was increased. Water was not only added as make-up fluid to the SFC effluent to ensure analyte focusing, but also as rinsing medium of the loops prior to the transfer of the fractions to the second dimension. In the SFC dimension, high efficiency and loadability were obtained by coupling two wide-bore columns (4.6 mm ID) in series. Evaporative light scattering (ELSD) and ultraviolet (UV) detection with standard and high-pressure flow cells were evaluated in terms of data acquisition speed and suppression of signal interferences originating from the supercritical carbon dioxide (CO₂) expansion.     

Two dimensional assisted liquid chromatography – a chemometric approach to improve accuracy and precision of quantitation in liquid chromatography using 2D separation,dual detectors,and multivariate curve resolution

Comprehensive two-dimensional liquid chromatography (LC × LC) is rapidly evolving as the preferred method for the analysis of complex biological samples owing to its much greater resolving power compared to conventional one-dimensional (1D-LC). While its enhanced resolving power makes this method appealing, it has been shown that the precision of quantitation in LC × LC is generally not as good as that obtained with 1D-LC. The poorer quantitative performance of LC × LC is due to several factors including but not limited to the undersampling of the first dimension and the dilution of analytes during transit from the first dimension (¹D) column to the second dimension (²D) column, and the larger relative background signals. A new strategy, 2D assisted liquid chromatography (2DALC), is presented here. 2DALC makes use of a diode array detector placed at the end of each column, producing both multivariate ¹D and two-dimensional (2D) chromatograms. The increased resolution of the analytes provided by the addition of a second dimension of separation enables the determination of analyte absorbance spectra from the ²D detector signal that are relatively pure and can be used to initiate the treatment of data from the first dimension detector using multivariate curve resolution–alternating least squares (MCR–ALS). In this way, the approach leverages the strengths of both separation methods in a single analysis: the ²D detector data is used to provide relatively pure analyte spectra to the MCR–ALS algorithm, and the final quantitative results are obtained from the resolved ¹D chromatograms, which has a much higher sampling rate and lower background signal than obtained in conventional single detector LC × LC, to obtain accurate and precise quantitative results. It is shown that 2DALC is superior to both single detector selective or comprehensive LC × LC and 1D-LC for quantitation of compounds that appear as severely overlapped peaks in the ¹D chromatogram – this is especially true in the case of untargeted analyses. We also anticipate that 2DALC will provide superior quantitation in targeted analyses in which unknown interfering compounds overlap with the targeted compound(s). When peaks are significantly overlapped in the first dimension, 2DALC can decrease the error of quantitation (i.e., improve the accuracy by up to 14-fold compared to 1D-LC and up to 3.8-fold compared to LC × LC with a single multivariate detector). The degree of improvement in performance varies depending upon the degree of peak overlap in each dimension and the selectivities of the spectra with respect to one another and the background, as well as the extent of analyte dilution prior to the ²D column.