Separation Of Complex Samples Research Articles

Two-dimensional strong cation-exchange liquid chromatography/reversed-phase pressurized capillary electrochromatography for separation of complex samples

A 2-D separation platform was constructed using micro strong cation-exchange liquid chromatography (μ-SCXLC) and reversed-phase pressurized capillary electrochromatography (RP-pCEC) for the analysis of complex samples. Samples were fractionated by the first-dimension μ-SCXLC with a linear solvent gradient and then injected into the second-dimension RP-pCEC for further separation. The μ-SCXLC/RP-pCEC 2-D system with three separation mechanisms, namely strong cation-exchange, reversed-phase chromatography and electrophoresis, provided high selectivity, high resolution and high peak capacity compared to one-dimensional chromatographic approaches. Separation effectiveness of this 2-D system was demonstrated by the analysis of different kinds of complex samples, such as traditional Chinese medicine Cortex Phellodendri, bovine serum albumin (BSA) tryptic digest and real serum tryptic digest. A theoretical peak capacity of approximately 1200 was achieved, which proves its promising potential for the separation and analysis of complex samples.

Ultra-High Pressure Liquid Chromatographic (UPLC) in Separation and Analysis of Botanicals and Natural Products: Retention Mechanism Studies

Liquid chromatography with C18-bonded packings and aqueous-organic eluents is a mature, ubiquitous, and incredibly efficient method for the separation and analysis of botanicals and natural products. Recent improvements in pump technology (1,200bar pressure) and silica chemistry (very stable, bridged-ethylene hybrids) have allowed the use of very small particles (<2µm) to achieve high efficiency separations of complex samples very rapidly. Despite the proven efficacy of UPLC, no overarching theory has emerged to describe the basic retention mechanism(s) operative in these complex chromatographic systems. The eluent usually consists of water with varying concentrations of an organic component, small concentrations of a polar acid, as well as buffers to control pH. The stationary phase often consists of a silica base with hydrophilic silanol and hydrophobic siloxane groups on the surface of the solid. The octadecane coating is chemically-bonded to the silica so one end is tethered and acts like a solid while the other end is free to act as a liquid. It is an unsettled issue whether the bonded octadecane on the silica exists in a collapsed state that supports adsorption or an extended (solvated) state that facilitates absorption of analytes. The eluent and stationary phases are complex enough by themselves. However, in a chromatographic column the stationary phase and eluent interact in dynamic equilibrium. In particular, almost all of the eluent components will be taken up in varying degrees by the stationary phase. It is this complex, in situ, dynamic equilibrium condition that is so difficult to model. The technique known as mass spectrometric tracer pulse chromatography1 was used to measure the uptake of eluent components by typical UPLC packing material. The ultimate goal is the determination of the effect of such eluent uptake on the retention of analytes. Unfortunately, it is impossible to directly measure the amount of anything absorbed by a liquid (C18) or adsorbed by a solid (silica or C18) from liquid eluents. However, the excess amount of eluent absorbed in or adsorbed on the stationary phase can be determined. An example of an excess isotherm of acetonitrile taken up by a C18-bonded BEH column as a function of eluent composition is given in Figure 1. The maximum absolute amount of acetonitrile taken up by the stationary phase can, however, be estimated from the isotherm data. It was found that for a UPLC column (2.1×150mm), the following amounts of eluent were either absorbed or adsorbed by the bonded stationary phase. The volume of bonded C18 material in the same column (˜40µL) can be estimated from the bonding density (3.1µmol/m2), the surface area (185m2/g) and the amount of packing in the column (˜300mg). Thus, the isotherm measurements indicate that the volume of ACN that acts as a part of the stationary phase (˜40µL) is essentially equal to the volume of octadecane chemically bonded to the packing material. The exact effect of this uptake of the retention of analytes is very difficult to isolate from the effects of the multiple other factors influencing the chromatographic performance of UPLC systems.

Tandem hollow-fiber flow field-flow fractionation

Reinjection of one ore more collected fractions of eluted samples is recognized as a useful procedure in analytical separation techniques, among which field-flow fractionation (FFF), to improve the actual separation of complex samples. Hollow-fiber flow FFF (HF5) is a micro-channel subset of flow FFF (F4), which has recently reached a performance comparable to that of standard, flat-channel F4. To further improve HF5 of complex protein samples, we present a new device and method for in-line, reinjection HF5 that we call tandem HF5 (HF5/HF5). HF5 is ideally suited for tandem operation because (1) small channel volume and low operation flow rates allow reducing dilution and volume of the collected fractions, and (2) the relaxation/focusing step that takes place between the 1st and 2nd run (refocusing) allows reestablishing the volume and concentration of the sample plug before the 2nd elution. HF5/HF5 proves particularly effective in the case of oligomeric proteins since it allows collecting and reinjecting the bands that correspond to each separated oligomeric form. This provides information on the dynamic equilibria between the different oligomers. For HF5/HF5 operations, a modified, prototype HF5 instrumentation is presented which includes a “trap” constituted of a four-port, two-way valve positioned downstream the UV detector and a collection loop. The effect of refocusing conditions on HF5/HF5 performance is investigated by varying refocusing time. With a complex protein samples such as blood serum, HF5/HF5 can improve detectability of the low abundance components since overloading effects due to high-abundance components are reduced. This is shown for serum lipoproteins: while after the 1st run high density lipoproteins (HDLs) are not separated from high-abundance serum proteins, after the 2nd run it is shown possible to separate the HDL subclasses.

Interface of two dimensional liquid chromatography

Two dimensional liquid chromatography (2D-LC) has become an important analytical tool for the separation of complex samples due to enhanced peak capacity and selectivity compared to one-dimensional LC. The interface of the two dimensions is the key technology in 2D-LC system. The interface shall provide mobile phase removal from the 1st D-LC and solutes focusing before the fraction transfer into the 2nd D-LC. A comprehensive review on the interface is presented, and 51 references are cited.

Ionic liquid‐bonded polysiloxane as stationary phase for capillary gas chromatography

Ionic liquid (IL) stationary phase is especially suitable for separation of complex samples, owing to the "dual nature" of IL. In this study, a synthetic method of ionic liquid-bonded polysiloxane (PSOIL) as stationary phase of GC was proposed. Then, the PSOIL was used to prepare an 8 m capillary column by static method. The column efficiency was measured to be about 4000 plates/m (k=3.55, naphthalene) after the column had been conditioned at 210°C. The durability of PSOIL column was better than that of the mixed stationary phase of IL and OV-1. Moreover, the Abraham solvation parameter model was employed to characterize the PSOIL. The result revealed that the PSOIL had stronger dispersion force (l) than neat IL and stronger hydrogen bond basicity (a) than DB-1. That meant the PSOIL might offer good selectivity for both polar and non-polar analytes. The column exhibited unique selectivity for various organic substances, such as the homologous compounds of alkanes, esters, alcohols and aromatic compounds. It was also found that some aromatic positional isomers could be separated better on the PSOIL column than on the DB-1 column. Furthermore, the stationary phase was suitable for separation of high-boiling point compounds such as polycyclic aromatic hydrocarbons, phthalic esters, etc. All of these demonstrated that the PSOIL offered good selectivity and high separation efficiency for a wide range of analytes.

Quantitation of the Ribosomal Protein Autoregulatory Network Using Mass Spectrometry

Relative levels of ribosomal proteins were quantified in crude cell lysates using mass spectrometry. A method for quantifying cellular protein levels using macromolecular standards is presented that does not require complex sample separation, identification of high-responding peptides, affinity purification, or postgrowth modifications. Perturbations in ribosomal protein levels by overexpression of individual proteins correlate to known autoregulatory mechanisms and extend the network of ribosomal protein regulation.

Recent advances in capillary electrochromatography and its coupling techniques

As a novel micro-separation technique, capillary electrochromatography (CEC) has the merits of high efficiency, high selectivity, high resolution and rapid analysis. However, the small-volume injection manipulated in capillary dimensions poses a great challenge for detectors in achieving high sensitivity. Currently, one of the major researches into CEC involves the development of some sensitive detection modes. The general introduction, which includes the historical perspectives and the principles of CEC, is briefly described. The recent advances about CEC coupled with various detectors and its applications in the separation of complex samples are summarized. A total of 141 references are reviewed.

“Click dipeptide”: A novel stationary phase applied in two-dimensional liquid chromatography

2D-HPLC is an important technique for the separation of complex samples. Developing new types of stationary phases is of great interest to construct 2D-LC systems with high orthogonality. In this study, a novel stationary phase-Click dipeptide ( l-phenylglycine dipeptide) was prepared by immobilization of α-azido l-phenylglycine dipeptide on alkyne-silica via click chemistry. In the preparation of this new material, an efficient, inexpensive and shelf-stable diazo transfer reagent (imidazole-1-sulfonyl azide hydrochloride) was utilized to transfer the amino group of l-phenylglycine to corresponding azido group under mild conditions. The Click dipeptide thus prepared was confirmed by FT-IR, solid state CP/MAS 13C NMR and elemental analysis. The Click dipeptide packing showed high orthogonality with C18, which reached 63.5%. An off-line 2D-RP/RPLC system was developed to analyze a traditional Chinese medicine (TCM)- Rheum Palmatum L. The results showed high orthogonality between Click dipeptide and C18 as well as great separating power in the practical separation of complex samples.

Fluorescein thiocarbamyl amino acids as internal standards for migration time correction in capillary sieving electrophoresis

A number of algorithms have been developed to correct for migration time drift in capillary electrophoresis. Those algorithms require identification of common components in each run. However, not all components may be present or resolved in separations of complex samples, which can confound attempts for alignment. This paper reports the use of fluorescein thiocarbamyl derivatives of amino acids as internal standards for alignment of 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ)-labeled proteins in capillary sieving electrophoresis. The fluorescein thiocarbamyl derivative of aspartic acid migrates before FQ-labeled proteins and the fluorescein thiocarbamyl derivative of arginine migrates after the FQ-labeled proteins. These compounds were used as internal standards to correct for variations in migration time over a two-week period in the separation of a cellular homogenate. The experimental conditions were deliberately manipulated by varying electric field and sample preparation conditions. Three components of the homogenate were used to evaluate the alignment efficiency. Before alignment, the average relative standard deviation in migration time for these components was 13.3%. After alignment, the average relative standard deviation in migration time for these components was reduced to 0.5%.

Computer-aided target optimization for traditional Chinese medicine by ultra-performance liquid chromatography

A new strategy of target optimization was developed for resolving the separation problems of complex samples. Computer-aided target optimization for separation of complex samples by ultra-performance liquid chromatography (UPLC) was studied. Taking traditional Chinese medicine (TCM) ( Rhizoma Coptidis) as an example, separation conditions of target compounds – overlapping peaks and all components – were optimized based on moved overlapping separation ranging map (OSRM) and artificially intervention. After calculating retention parameters and peak shape parameters of the target peaks, the optimization was operated in an emulational picture through the computer without further experiments. The emulational chromatogram was proved to be authentic by the estimated values, which were almost identical to experimental results. The method was very helpful for obtaining the satisfied separation conditions of target compounds rapidly and efficiently.

“Click oligo(ethylene glycol)”: An excellent orthogonal stationary phase to C18 for two-dimensional reversed-phase/reversed-phase liquid chromatography

A novel click oligo(ethylene glycol) (Click OEG) stationary phase was proposed for constructing two-dimensional reversed-phase/reversed-phase liquid chromatography system (2D-RP/RPLC) combined with C18. The new stationary phase was prepared by immobilization of oligo(ethylene glycol) on silica support via click chemistry. And the resulting material was characterized by FT-IR, solid state CP/MAS 13C NMR and elemental analysis. The investigation on orthogonality between Click OEG and C18 was performed by geometric approach with a set of test solutes. The orthogonality reached 68.94%, which is much higher than other combinations. An offline 2DLC system consisted of Click OEG and C18 was constructed and applied to the analysis of a kind of traditional Chinese medicine Lignum Dalbergiae Odoriferae. Excellent separation results validated the high orthogonality between the Click OEG and C18 in the practical separation of complex samples.

Simultaneous separation of hydrophilic and hydrophobic compounds by using an online HILIC‐RPLC system with two detectors

In the present study, we developed a novel online hydrophilic interaction chromatography (HILIC)-RPLC separation system for simultaneous separation of both hydrophilic and hydrophobic solutes in a complex sample with one injection. A HILIC Si column and a C18 column were hyphenated with an interface including two electronic 2-position valves, a solvent pump, and a solute transfer column. By using column-switching technique, the nonretained hydrophobic solutes were eluted out of the HILIC column and transferred into the C18 column to perform further separation. The hydrophilic solutes were separated on the HILIC column at the same time. One detector was equipped for each column to record the individual chromatograms. By separating a standard mixture and a traditional Chinese medicine (TCM) extract, the developed HILIC-RPLC system demonstrates its potential for "entire-components" separation of complex samples with improved peak capacity and throughput compared with the common single-column LC.

Stationary phases for hydrophilic interaction chromatography, their characterization and implementation into multidimensional chromatography concepts

Hydrophilic interaction chromatography (HILIC) is becoming increasingly popular for separation of polar samples on polar columns in aqueous-organic mobile phases rich in organic solvents (usually ACN). Silica gel with decreased surface concentration of silanol groups, or with chemically bonded amino-, amido-, cyano-, carbamate-, diol-, polyol-, or zwitterionic sulfobetaine ligands are used as the stationary phases for HILIC separations, in addition to the original poly(2-sulphoethyl aspartamide) strong cation-exchange HILIC material. The type of the stationary and the composition of the mobile phase play important roles in the mixed-mode HILIC retention mechanism and can be flexibly tuned to suit specific separation problems. Because of excellent mobile phase compatibility and complementary selectivity to RP chromatography, HILIC is ideally suited for highly orthogonal 2-D LC-LC separations of complex samples containing polar compounds, such as peptides, proteins, oligosaccharides, drugs, metabolites and natural compounds. This review attempts to present an overview of the HILIC separation systems, possibilities for their characterization and emerging HILIC applications in 2-D off-line and on-line LC-LC separations of various samples, in combination with RP and other separation modes.

Biomolecular structural separations by ion mobility–mass spectrometry

Following the paradigm of the human genome project, much of current systems biology research entails characterizing, quantifying, and cataloging the biomolecular inventory of a sample at specific dimensions of space (e.g., cellular, tissue, or organism levels) and time (e.g., point in the life cycle, healthy vs. diseased state). These largely hypothesis-independent data are then integrated with the development of bioinformatic algorithms to derive an understanding of how different molecular networks interact and function, which subsequently provide a framework for performing hypothesis-driven studies [1]. This reductionist approach (i.e., measuring gene and protein expression levels, evaluating protein post-translational modifications, metabolite levels, etc.) is necessary to begin parameterizing the extraordinarily complex molecular interaction networks that regulate, control, and perturb biochemical processes, rather than directly analyzing the biological entity in its entirety. Quantitation of gene transcription is dominated by the use of gene arrays, while protein expression and metabolite levels are commonly measured using mass spectrometry (MS) or chromatography separations coupled with MS-based techniques [2]. Through a combination of measurements, specific biomolecular networks and molecular signatures of disease-states can be elucidated. To illustrate the scale of such studies, ca. 10 and 10 measurements were performed to generate a perturbation model of galactose metabolism in yeast and to determine a suite of prostate cancer biomarkers, respectively [2, 3]. Such large-scale experiments motivate the development of measurement strategies that incorporate higher throughput, higher selectivity, are comprehensive, and require minimal sample manipulation. Emerging developments in two-dimensional gas-phase separations on the basis of ion mobility–mass spectrometry (IM–MS) have demonstrated great promise for biomolecular separations of complex samples. This trend report focuses on several of the unique capabilities of IM–MS in contrast with MS-only strategies and provides a description of several key measurement areas in which IM–MS will likely play an increasing role in the near future. Detailed discussions of IM–MS techniques for biomolecular applications are provided elsewhere [4–9].

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 × 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 × 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 × 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 60 s. 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.

How to generate peak capacity in column liquid chromatography: The Halász nomograms revised

For the separation of complex samples it is necessary to run the analyses on a chromatographic set-up with high peak capacity. The concept of peak capacity is broader than the one of the theoretical plate numbers because it covers both the column characteristics and the run time of the separation. Therefore, a desired peak capacity can be obtained with many combinations of particle diameter, column length, pressure, and analysis time: either with a short column, small-diameter packing, short analysis time, and high pressure; or with less pressure at the expense of a longer column, larger-diameter packing, and longer analysis time. These combinations can be presented in the form of nomograms with the analysis time as x-axis and the peak capacity as y-axis and including particle diameter, column length, and pressure as parameters. The practical limits of the peak capacity are given by the maximum pressure delivered by the pump in use. These considerations are valid for isocratic and gradient separations as well. They are based on a 1982 paper by Halász and Görlitz and apply the concept of HPLC columns used at their Van Deemter optimum.

High‐Throughput Approach to 2‐Dimensional Fingerprinting of Complex Mixtures such as Beverages

A combination of two or more analytically orthogonal separation modes not only multiplies their differentiation powers but may reveal some new relationships in complex molecular distributions, thus providing analytical information that individual separation dimensions could not deliver. The resulting multidimensional chromatogram may serve as a very sensitive differentiator of complex mixtures. Such a “fingerprint” can be linked to certain application relevant characteristics that are difficult to measure directly, including sensory parameters like taste and smell. Many traditional fields of industry, such as food, chemicals, and materials, could benefit from the power of multidimensional separations. However, lack of ready to go instrumental solutions, long analysis times, and overwhelming data streams, contribute to the relatively slow penetration of the 2‐D HPLC and similar separation technology into the industrial and routine analysis labs. In this paper, we report on a high‐throughput and low cost per analysis approach to the two‐dimensional separations of complex samples. It is based on the ion‐exchange fractionation using the 96‐well plate followed by the reversed‐phase HPLC profiling by an eight channel capillary HPLC instrument. Fingerprinting of common beverages is shown and discussed as an example, with a much broader applicability in mind.

Study on Gender Difference Based on Metabolites in Urine by Ultra High Performance Liquid Chromatography/Time of Flight Mass Spectrometry

Metabolites in urine can illustrate the physical condition of an individual as a whole. Ultra high performance liquid chromatography/time of flight mass spectrometry (UPLC/TOF-MS) is a relative new technique for the separation of complex samples. The aim of this study is to assess the feasibility of metabonomics in gender difference in unrestricted conditions, i.e. for healthy volunteers there are no strict controls such as food, life style and the collection of urine samples. In this work, 31 spontaneous urine samples were collected and analyzed by using UPLC/TOF-MS. Principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA) models were tested and compared in samples classification. The gender discrimination was highly improved and some gender related biomarkers were found by PLS-DA. These preliminary results suggested that UPLC/MS-based approaches coupled with pattern recognition show promise for metabonomics.

A principal component analysis based method to discover chemical differences in comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC-TOFMS) separations of metabolites in plant samples

Comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC × GC-TOFMS) provides high resolution separations of complex samples with a mass spectrum at every point in the separation space. The large volumes of multidimensional data obtained by GC × GC-TOFMS analysis are analyzed using a principal component analysis (PCA) method described herein to quickly and objectively discover differences between complex samples. In this work, we submitted 54 chromatograms to PCA to automatically compare the metabolite profiles of three different species of plants, namely basil ( Ocimum basilicum), peppermint ( Mentha piperita), and sweet herb stevia ( Stevia rebaudiana), where there were 18 chromatograms for each type of plant. The 54 scores of the m/ z 73 data set clustered in three groups according to the three types of plants. Principal component 1 (PC 1) separated the stevia cluster from the basil and peppermint clusters, capturing 61.84% of the total variance. Principal component 2 (PC 2) separated the basil cluster from the peppermint cluster, capturing 16.78% of the total variance. The PCA method revealed that relative abundances of amino acids, carboxylic acids, and carbohydrates were responsible for differentiating the three plants. A brief list of the 16 most significant metabolites is reported. After PCA, the 54 scores of the m/ z 217 data set clustered in three groups according to the three types of plants, as well, yielding highly loaded variables corresponding with chemical differences between plants that were complementary to the m/ z 73 information. The PCA data mining method is applicable to all of the monitored selective mass channels, utilizing all of the collected data, to discover unknown differences in complex sample profiles.

A practical approach to maximizing peak capacity by using long columns packed with pellicular stationary phases for proteomic research

Maximization of peak capacity is a very important step in developing one-dimensional separations of complex samples. In recent work, it was shown that the use of small particles in combination with the new technique of ultrahigh pressure liquid chromatography (UHPLC) was able to generate very high peak capacities. Here we show the ability of conventional HPLC instrumentation to give comparable peak capacities to those obtained in UHPLC for the important case of complex mixtures of peptides but at much lower pressures by using a 60 cm long set of columns packed with 5 μm pellicular (superficially porous) particles. We first show, in complete agreement with the well known results of the theory of isocratic separations that, when time is not limiting, the best peak capacities in gradient elution chromatography are obtained by using large particles and the longest column that can be operated at the pump's pressure limit. Two different types of 5 μm particles (superficially porous and totally porous) were compared for their efficiency in gradient chromatography of peptides. We find that the pellicular material gave about 50% higher peak capacity compared to the analogous porous material. A 60 cm column set packed with pellicular particles was made by connecting short columns in series; a peak capacity of about 460 was obtained in 4 h at room temperature. Increasing the column temperature to 70 °C reduced the analysis time to 2 h and further increased the peak capacity to more than 500. The number of peaks observed in the separation of bovine serum albumin tryptic peptides was greatly increased and the separation quality was significantly improved.