Targeted multidimensional gas chromatography using microswitching and cryogenic modulation

A new method is described that allows fast target analysis in multidimensional gas chromatography by using a microswitching valve between two GC columns, with cryogenic trapping and rapid re-injection of trapped solutes in the second dimension. The essence of the procedure is that heart-cut fractions from the first column (1D) can be selectively transferred to column 2 (2D), where a moveable cryogenic trap first focuses the transferred solute(s) at the head of the second column and then permits their facile rapid analysis on 2D. Since 2D is a short narrow-bore column, which exhibits very fast analysis (on the order of a few seconds elution), peak responses (heights) are significantly enhanced (by up to 40-fold). Additionally, by using a 2D phase of a selectivity different from that used for 1D, it is possible to also separate components that are not resolved on the first column and to increase the resolution for other compounds. The heart-cut valve isolates the section(s) of solutes of interest from the first column separation, and this provides a considerable simplification to the chromatogram-in addition to the separation and sensitivity advantages. By using this method, multidimensional gas chromatography with multiple heart-cuts can be completed within the same time as the primary column separation. Since the described method permits non-heart-cut fractions to be transferred to a monitor detector, normal detection of these fractions is still permitted. By modulation of the cryotrap, it is also possible to achieve comprehensive two-dimensional gas chromatography for the heart-cut fractions; however, only those compounds passed to the second, separation column, which passes through the cryotrap, will be subjected to GC x GC analysis. The technique and the various modes of operation are described in this paper.     

Chromatographic characteristics of surfactant-mediated separations: micellar liquid chromatography vs ion pair chromatography.

The effects of concentrations of organic solvent and surfactant on elution strength and selectivity in MLC and IPC are studied. It is observed that selectivity between most pairs of solutes used in this study increases in MLC and either decreases or passes through a minimum in IPC, with the volume fraction of organic modifier. In both MLC and IPC, selectivity varies with surfactant concentration; however, the overall variation in selectivity and elution order are more pronounced in MLC. The solvent strength decreases in IPC and increases in MLC as a result of an increase in surfactant concentration. An iterative regression design is used to predict the optimum mobile-phase compositions in terms of solvent strength and selectivity. The correlation between the predicted and measured chromatograms is excellent in MLC and poor in IPC. This is due to a more regular and reproducible retention behavior in MLC which greatly facilitates the development of robust methodologies. For a mixture of amino acids and peptides, a large retention gap between the first and the last eluting solutes is observed in IPC, which makes the use of organic solvent gradient inevitable. However, a better separation for the same mixture of solutes can be achieved in MLC isocratically. Apparently, the general elution problem can be alleviated in MLC by using an optimum eluent composition. It is observed that the efficiencies of MLC and IPC are comparable. The above observations indicate that MLC can be a powerful alternative to IPC in order to achieve optimized separations in shorter analysis time.     

An inductively coupled plasma carbon emission detector for aqueous carbohydrate separations by liquid chromatography

An inductively coupled plasma atomic emission spectrometer is used to detect carbon-containing compounds following separation by high-performance liquid chromatography. A calcium form ligand exchange column with distilled and deionized water as the mobile phase is used to separate carbohydrates. The eluting species are detected by monitoring the carbon atomic emission line at 193.09 nm. The mass detection limits using a photomultiplier tube for sucrose and glucose are 50 ng, while that for fructose is 60 ng. The carbon emission detector should provide the same detection limit for any compound with a similar mass percent of carbon, whether or not the compound exhibits appreciable absorption characteristics. While the carbon emission detector will universally detect any organic compound, it will discriminate against species with high molar absorptivity that may be present at low concentration. Such species may act as interferences in chromatograms generated with conventional UV-visible absorption detectors. To demonstrate the utility of the carbon emission detector, three sugars (glucose, fructose, sucrose) are determined in apple, crangrape, and orange juice.     

Pressure-driven reverse-phase liquid chromatography separations in ordered nonporous pillar array columns

Building upon the micromachined column idea proposed by the group of Regnier in 1998, we report on the first high-resolution reversed-phase separations in micromachined pillar array columns under pressure-driven LC conditions. A three component mixture could be separated in 3 s using arrays of nonporous silicon pillars with a diameter of approximately 4.3 microm and an external porosity of 55%. Under slightly retained component conditions (retention factor k' = 0.65-1.2), plate heights of about H = 4 microm were obtained at a mobile phase velocity around u = 0.5 mm/s. In reduced terms, such plate heights are as low as hmin = 1. Also, since the flow resistance of the column is much smaller than in a packed column (mainly because of the higher external porosity of the pillar array), the separation impedance of the array was as small as E = 150, i.e., of the same order as the best currently existing monolithic columns. At pH = 3, yielding very low retention factors (k' = 0.13 and 0.23), plate heights as low as H = 2 microm were realized, yielding a separation of the three component mixture with an efficiency of N = 4000-5000 plates over a column length of 1 cm. At higher retention factors, significantly larger plate heights were obtained. More experimental work is needed to investigate this more in depth. The study is completed with a discussion of the performance limits of the pillar array column concept in the frame of the current state-of-the-art in microfabrication precision.     

On-line coupling of supercritical fluid extraction with multidimensional microcolumn liquid chromatography/gas chromatography.

An on-line multidimensional supercritical fluid extraction/microcolumn liquid chromatography/capillary gas chromatography system (SFE/LC/GC) has been developed and applied to the quantitative determination of trace levels (parts per billion) of chlorpyrifos insecticide in grass field samples. This system provides all the advantages of an on-line multidimensional system, including increased resolving power, high sensitivity, quantitation, precision, and automation potential. Off-line analysis of the grass extracts by GC with an electron capture detector yielded a complex chromatogram from which it was difficult to quantitate the chlorpyrifos, but analysis of the extract by LC/GC yielded a simple chromatogram from which chlorpyrifos could be quantitated. On-line SFE/LC/GC resulted in reduced sample preparation with the grass extract being deposited directly on the LC microcolumn via an impactor interface, followed by the LC/GC separation. The reproducibility of the on-line SFE/LC/GC procedure was studied and found to yield a relative standard deviation of 10.8% for the determination of chlorpyrifos insecticide in grass field samples at a concentration of 160 ng/g. Using this method, the entire analysis including extraction, clean-up, and gas chromatography required less than 0.1 mL of organic solvent.     

A multidimensional differential proteomic platform using dual-phase ion-exchange chromatography-polyacrylamide gel electrophoresis/reversed-phase liquid chromatography tandem mass spectrometry

Differential proteomic analysis has arisen as a large-scale means to discern proteome-wide changes upon treatment, injury, or disease. Tandem protein separation methods are required for large-scale differential proteomic analysis. Here, a novel multidimensional platform for resolving and differentially analyzing complex biological samples is presented. The platform, collectively termed CAX-PAGE/RPLC-MSMS, combines biphasic ion-exchange chromatography with polyacrylamide gel electrophoresis for protein separation, quantification, and differential band targeting, followed by capillary reversed-phase liquid chromatography and data-dependent tandem mass spectrometry for quantitative and qualitative peptide analysis. CAX-PAGE provides high protein resolving power with a theoretical peak capacity of 3570, extendable to 7600, a wide protein mass range verified from 16 to 273 kDa, and reproducible differential sample comparison without the added expense of fluorescent dyes and imaging equipment. Demonstrated using a neuroproteomic model, CAX-PAGE revealed an increased number of differential proteins, 137, compared with 82 found by 2D difference gel electrophoresis. When combined with RPLC-MSMS for protein identification, an additional quantification step is performed for internal validation, confirming a 2-fold or greater change in 89% of identified differential targets.     

Chiral separations performed by enhanced-fluidity liquid chromatography on a macrocyclic antibiotic chiral stationary phase

The use of enhanced-fluidity liquid chromatography (EFLC) for chiral separations was demonstrated on a macrocyclic antibiotic column, Chirobiotic-V. This technique was compared to high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) for the separation of chiral compounds in normal-phase mode. The highest resolution was always observed for EFLC condition. Higher efficiency and shorter retention time were also observed for most separations with portions of CO(2) in the range of 0-50 mol %. Larger amounts of CO(2) caused efficiency to decrease and retention time to be prolonged. For some separations, the temperature was elevated to bring the mobile phase to the supercritical condition. Improved efficiency was obtained in SFC, whereas resolution and selectivity were worse. The use of EFLC in reversed-phase chiral separations was also tested. Enantiomer resolution improved under the EFLC condition. For the tested methanol/H(2)O mixture, fluoroform provided more significant improvements in chromatographic performance than CO(2) when used as a fluidity enhancing liquid. The use of EFLC instead of HPLC also caused a markedly lower pressure drop across the column for commonly used flow rates. The low-pressure drop will allow the use of longer columns or multiple columns to increase the total efficiency of the separation. Since chiral columns are often inefficient, this attribute may be very important for chiral separations.     

On-chip coupling of isoelectric focusing and free solution electrophoresis for multidimensional separations

We have developed an acrylic microfluidic device that sequentially couples liquid-phase isoelectric focusing (IEF) and free solution capillary electrophoresis (CE). Rapid separation (<1 min) and preconcentration (73x) of species were achieved in the initial IEF dimension. Using full-field fluorescence imaging, we observed nondispersive mobilization velocities on the order of 20 microm/s during characterization of the IEF step. This transport behavior allowed controlled electrokinetic mobilization of focused sample bands to a channel junction, where voltage switching was used to repeatedly inject effluent from the IEF dimension into an ampholyte-based CE separation. This second dimension was capable of analyzing all fluid volumes of interest from the IEF dimension, as IEF was 'parked' during each CE analysis and refocused prior to additional CE analyses. Investigation of each dimension of the integrated system showed time-dependent species displacement and band-broadening behavior consistent with IEF and CE, respectively. The peak capacity of the 2D system was approximately 1300. A comprehensive 2D analysis of a fluid volume spanning 15% of the total IEF channel length was completed in less than 5 min.     

Optimizing TiO2-based phosphopeptide enrichment for automated multidimensional liquid chromatography coupled to tandem mass spectrometry

An automated online multidimensional liquid chromatography system coupled to ESI-based tandem mass spectrometry was used to assess the effectiveness of TiO2 in the enrichment of phosphopeptides from tryptic digests of protein mixtures. By monitoring the enrichment of phosphopeptides, an optimized set of loading, wash, and elution conditions were realized for TiO2. A comparison of TiO2 with other resins used for phosphopeptide enrichment, Fe(III)-IMAC and ZrO2, was also carried out using tryptic digests of both simple and moderately complex protein mixtures; where TiO2 was shown to be superior in performance.     

Comprehensive two-dimensional normal-phase (adsorption)-reversed-phase liquid chromatography

A comprehensive two-dimensional HPLC system has been developed. It is based on the use of a microbore silica column operated in normal-phase (adsorption) mode (NP) in the first dimension and a monolithic type C18 column operated in reversed-phase (RP) mode in the second dimension. The interface was a 10-port, 2-position valve equipped with two storage loops. The first column was operated at a flow rate of 20 microL/min in isocratic mode, while the monolithic column flow rate was 4 mL/min and was operated in gradient mode. The sample loops had a volume of 20 microL each, and the analysis time in the second dimension was 1 min. In this way, every fraction from the first dimension was transferred on-line to the second dimension switching the automated valve every minute. A photodiode array detector has been used after the secondary column. The use of normal- and reversed-phase mode in the two dimensions can be helpful in the separation of complex mixtures of a natural origin that contain uncharged molecules of comparable dimension, different in polarity and hydrophobicity. The use of a microbore column in the first dimension permits the injection of a small volume in the secondary column, making the transfer of incompatible solvents from the first to the second dimension possible. Since the mobile phase in the NP separation is always stronger than the mobile phase at the head of the secondary column operated in RP mode, the initial eluent strength is important in order to obtain an effective focusing of the sample. The use of a monolithic type column in the second dimension permits the performance of very fast analysis operating at higher flow rates without loss of resolution, due to a higher permeability and increased mass-transfer properties in comparison to conventional particulate columns. Due to the brief reconditioning time necessary for monolithic columns, repetitive gradients can be carried out, extending the field of application to mixtures that contain components with different polarities. The utility of the system has been demonstrated in the analysis of the oxygen heterocyclic fraction of cold-pressed lemon oil, made up of coumarins and psoralens. These components may contain hydroxyl, methoxyl, isopentenyl, isopentenyloxyl, and geranyloxyl groups and oxygen-containing modification of the terpenoid side-chain groups, such as epoxides or vicinal diol groups. The relative location of the components in the 2D plane varied in relation to their chemical structure and allowed positive peak identification. The UV spectra recorded with the photodiode array detector supplied additional information that was used for the characterization of the studied sample.     

Electrogenerated chemiluminescence derivatization reagents for carboxylic acids and amines in high-performance liquid chromatography using tris(2,2'-bipyridine)ruthenium(II)

2-(2-Aminoethyl)-1-methylpyrrolidine and N-(3-aminopropyl)pyrrolidine (NAPP) were found to be selective and sensitive derivatization reagents for carboxylic acid by high-performance liquid chromatography (HPLC) with electrogenerated chemiluminescence detection using tris(2,2'-bipyridine)ruthenium(II). Free fatty acids and ibuprofen were used as model compounds of carboxylic acids, and the derivatization conditions were optimized with myristic acid as a representative of free fatty acids. All the fatty acids tested were reacted with NAPP to produce highly sensitive derivatives under the mild reaction conditions of room temperature for 30 min in acetonitrile containing 2-bromo-1-ethylpyridinium tetrafluoroborate and 9-methyl-3,4-dihydro-2H-pyrido[1,2-a]pyrimidin-2-one. The chemiluminescence intensities were similar for all fatty acids. The derivatives obtained from 10 free fatty acids were completely separated by reversed-phase chromatography under isocratic elution conditions. The on-column detection limits (signal-to-noise ratio of 3) with proposed HPLC separation and chemiluminescence detection were 70 and 45 fmol for myristic acid and ibuprofen, respectively. The free fatty acids in human plasma were successfully determined using the present method. Histamine, a model compound of primary amines, was also determined after precolumn derivatization with 3-(diethylamino)propionic acid at room temperature for 60 min in acetonitrile containing N,N'-dicyclohexylcarbodiimide and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine with the detection limit of 70 fmol.     

Near-lossless continuous phase modulation using the analog switching mode (V-shaped switching) in ferroelectric liquid crystals

The analog switching mode in ferroelectric liquid crystals, sometimes referred to as 'V-shaped switching,' has, thanks to its submillisecond switching capability, attracted much interest for future fast electro-optic displays where it is to be used for amplitude modulation. We have studied this mode for analog phase-only modulation. As V-shaped switching is based on a conical motion of the index ellipsoid this presents a challenging problem since both the orientation of the slow and fast axes, as well as the amount of birefringence varies in the switching process. We show theoretically, partly by means of Poincaré sphere analysis, that it is in fact possible to obtain near-lossless analog phase modulation between zero and pi radians in an ideal V-shaped switching cell through careful tuning of the polarization state of the input light. Furthermore, we were able to demonstrate this experimentally in a fabricated cell. Although this cell deviated slightly from the ideal conditions, e.g., the tilt cone half-angle was 38 degrees instead of the desired 45 degrees , we still obtained a continuous phase modulation between zero and 0.78pi rad with less than 2% modulation of the amplitude; the measured values agree very well with our numerical simulations of the real device.     

Anion and cation mixed-bed ion exchange for enhanced multidimensional separations of peptides and phosphopeptides

Shotgun proteomics typically uses multidimensional LC/MS/MS analysis of enzymatically digested proteins, where strong cation-exchange (SCX) and reversed-phase (RP) separations are coupled to increase the separation power and dynamic range of analysis. Here we report an on-line multidimensional LC method using an anion- and cation-exchange mixed bed for the first separation dimension. The mixed-bed ion-exchange resin improved peptide recovery over SCX resins alone and showed better orthogonality to RP separations in two-dimensional separations. The Donnan effect, which was enhanced by the introduction of fixed opposite charges in one column, is proposed as the mechanism responsible for improved peptide recovery by producing higher fluxes of salt cations and lower populations of salt anions proximal to the SCX phase. An increase in orthogonality was achieved by a combination of increased retention for acidic peptides and moderately reduced retention of neutral to basic peptides by the added anion-exchange resin. The combination of these effects led to approximately 100% increase in the number of identified peptides from an analysis of a tryptic digest of a yeast whole cell lysate. The application of the method to phosphopeptide-enriched samples increased by 94% phosphopeptide identifications over SCX alone. The lower pKa of phosphopeptides led to specific enrichment in a single salt step resolving acidic phosphopeptides from other phospho- and non-phosphopeptides. Unlike previous methods that use anion exchange to alter selectivity or enrich phosphopeptides, the proposed format is unique in that it works with typical acidic buffer systems used in electrospray ionization, making it feasible for online multidimensional LC/MS/MS applications.     

Computer simulation (based on a linear-elution-strength approximation) as an aid for optimizing separations by programmed-temperature gas chromatography

If the dependence of retention on temperature is specified for the various components of a sample in isothermal gas chromatography (GC), it is possible to predict retention, bandwidth, and resolution for programmed-temperature GC separations as a function of experimental conditions. The use of a linear-elution-strength (LES) approximation for isothermal retention allows these predictions to be carried out more easily and conveniently, in turn facilitating rapid simulations with a personal computer. This approach to GC method development appears promising, especially if segmented-temperature programs are used. The LES approximation also provides added insight into how different factors affect separation in programmed-temperature GC.     

High-throughput screening for enzyme inhibitors using frontal affinity chromatography with liquid chromatography and mass spectrometry

This work presents new frontal affinity chromatography (FAC) methodologies for high-throughput screening of compound libraries, designed to increase screening rates and improve sensitivity and ruggedness in performance. A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented in the identification of potential enzyme inhibitors from two libraries of trisaccharides. Effluent from the FAC column was fractionated, sequentially processed via LC/MS, and referenced to a similar analysis through a control FAC column lacking the enzyme. The resulting multidimensional data sets were compared across corresponding sample and control fractions to identify binders, in a semiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first library of 81 compounds, exhibiting an estimated Kd value of 0.3 microM. Other binders yielded Kd values ranging from 0.35 to 3.35 microM. To demonstrate the improvement in performance of this FAC-LC/MS approach over the conventional online FAC/MS approach, 15 compounds from this library were blended with a second library of 1000 synthetic trisaccharides and screened against GnT-V. All ligands in the 15-compound set were identified in this larger screen, and no ligands of greater affinity than compound 1 were found. Our results show that FAC-LC/MS is a reliable method for screening large compound libraries directly and useful for large-scale ligand discovery initiatives.     

Characterization of capillary-channeled polymer fiber stationary phases for high-performance liquid chromatography protein separations: Comparative analysis with a packed-bed column

Capillary-channeled polymer (C-CP) fibers are investigated as reversed-phase (RP) stationary phases for high-performance liquid chromatography of proteins. A comparative analysis of column characteristics for polypropylene and poly(ethylene terephthalate) C-CP fiber columns and a conventional packed-bed (C4-derivatized silica) column has been undertaken. Five proteins (ribonuclease A, cytochrome c, lysozyme, myoglobin, bovine serum albumin) were used to investigate the separation characteristics under typical RP gradient conditions. Column performance was compared under standard (identical) and optimized RP chromatographic conditions. The gradient compositions utilized with the C-CP fiber columns are similar to those used with conventional columns, employing flow rates in the 1-6 mL/min range and gradient rates of approximately 1%/min. The packed-bed column was operated as prescribed by the column manufacturer. The retention factor (k'), separation factor (alpha), resolution (Rs), asymmetry factor (As), elution order, and peak capacity values of a four protein separations performed on the C-CP fiber columns are compared to the same separation on the C4 column. One unique feature observed here is the lessening of the percentage of organic modifier necessary to elute the proteins from the fiber phases with increased linear velocity. The potential contribution of the different stationary phases to protein denaturation was evaluated through a spectrophotometric enzymatic activity assay. The repeatability of retention times under both sets of conditions for six consecutive injections of lysozyme on each C-CP fiber column is < or =1.5% RSD. The column-to-column reproducibility of retention times for three columns of each fiber type is also < or =1.5% RSD. The overall performance of the C-CP fiber columns was comparable to the conventional column used in these studies. Basic characteristics demonstrated here suggested further developments in the areas of ultrafast protein separations and preparative-scale protein chromatography.     

Capillary ultrahigh performance liquid chromatography with elevated temperature for sub-one minute separations of basal serotonin in submicroliter brain microdialysate samples

Improving the time resolution in microdialysis coupled to high performance liquid chromatography (HPLC) requires that the volume of the separation system be decreased. A low-volume separation permits smaller microdialysate volumes to be injected without suffering a sensitivity loss from dilution. Thus, improved time resolution can be achieved with offline analysis simply by decreasing the separations system volume. For online (near real-time) analysis, there is a further requirement. The separation speed must be at least as fast as the sampling time. Here, the combined use of high column pressures and temperatures, sub-2-μm stationary phase particles, capillary columns, and sensitive, low dead-volume detection resulted in a retention time for the neurotransmitter serotonin of less than 1 min in a 500 nL dialysate sample volume. Two sensitive detectors, photoluminescence following electron transfer (PFET) and electrochemical, were used for the detection of subnanomolar concentrations of serotonin in brain microdialysate samples. The general principles developed are applicable to a wide range of separations with the additional advantages of increases in sample throughput and decreases in mobile phase usage.     

Evaluation of the deuterium isotope effect in zwitterionic hydrophilic interaction liquid chromatography separations for implementation in a quantitative proteomic approach

Quantitative methodologies for the global in-depth comparison of proteomes are frequently based on chemical derivatization of peptides with isotopically distinguishable labeling agents. In the present work, we set out to study the feasibility of the dimethyl labeling method in combination with ZIC-cHILIC (zwitterionic hydrophilic interaction liquid chromatography) technology for quantitative proteomics. We first addressed the potential issue of isotope effects perturbing the essential coelution of differently labeled peptides under ZIC-cHILIC separation. The deuterium incorporation-induced effect can be largely eliminated by favoring the mixed-mode ZIC-cHILIC separation based on combined hydrophilic and ionic interactions. Then, we evaluated the performance and applicability of this strategy using a sample consisting of human cell lysate. We demonstrate that our approach is suitable to perform unbiased quantitative proteome analysis, still quantifying more than 2500 proteins when analyzing only a few micrograms of sample.