An automated orthogonal two-dimensional liquid chromatograph

A simple approach to two-dimensional liquid chromatography has been developed by coupling columns of different selectivity using a 12-port, dual-position valve and a standard HPLC system. The valve at the junction of the two columns enables continuous, periodic sampling (injection) of the primary column eluent onto the secondary column. The separation in the primary dimension is comparable to conventional HPLC, whereas the secondary column separation is fast, lasting several seconds. The high-speed separation in the secondary dimension enables the primary column eluent to be sampled with fidelity onto the secondary column throughout the chromatographic run. One might expect a coupled column liquid chromatography system operating in reverse-phase mode to be strongly correlated and, hence, inefficient. However, by applying a solvent gradient in the primary dimension and by progressively incrementing the solvent strength in the secondary dimension (tuning), the inefficiency or cross correlation between the two dimensions is minimized. In a tuned two-dimensional system, the influence of primary column retention (usually hydrophobicity) is minimal on secondary column retention. This enables subtle differences in component interaction with the two stationary phases to dominate the secondary column retention. The peaks are randomly dispersed over a retention plane rather than along a diagonal, resulting in an orthogonal separation. The peak capacity is multiplicative, and each component has a unique pair of retention times, enabling positive identification. In addition, the location of the component provides two independent measures of molecular properties. The 2D-LC system was evaluated by analyzing a test mixture made of some aromatic amines and non-amines on different secondary columns (ODS-AQ/ODS monolith, ODS/amino, ODS/cyano). The relative location of sample components in the two-dimensional plane varied significantly with change in secondary column. Among the secondary columns, the amino and cyano columns offered the most complementary separation, with the retention order of several components reversed in the secondary dimension. The theoretical peak capacity of the 2D-LC system was around 450 for a separation lasting 30 min. A 2D-LC system involving amino and cyano columns resulted in a high-speed separation of the test mixture, with most of the chemical components resolved within a few minutes.     

Separation of barbiturates and phenylthiohydantoin amino acids using the thermally tuned tandem column concept

There are many more choices of column type than of eluent type for method development in reversed-phase liquid chromatography. It is common to switch between different column types or between the same type from different suppliers to achieve the desired separations. The key difficulty in modulating band spacing by adjusting the column type is that it is a discontinuous, "hit or miss" proposition. The thermally tuned tandem column (T3C) concept effectively solves this problem by connecting two columns in series and independently controlling the two column temperatures. The columns are chosen to have distinctively different chromatographic selectivities (band spacing), so that the unresolved peaks on one column are separated by the other. The optimized separation in the T3C is achieved by simultaneously tuning the two column temperatures. In this study, we used the T3C combination of a carbon and a conventional bonded phase for the separation of barbiturates and phenylthiohydantoin amino acids (PTH-amino acids). Good peak shapes and comparable retention times were observed on the two phases at room temperature. The selectivities on the two phases are quite different. Baseline separations were easily achieved with the T3C set although neither column could individually resolve all the peaks. We further compared the separation of barbiturates optimized by the T3C approach with that optimized by adjusting the mobile phase. We found that T3C gave a better separation. We believe that the T3C combination of a carbon phase and a bonded conventional reversed-phase material provides a powerful and general method to optimize the separation of various mixtures.     

A New HPLC Approach for the Determination of Hydrophilic and Hydrophobic Components: The Case of Pseudoephedrine Sulfate and Loratadine in Tablets

Effective isocratic separations of decongestants and antihistamines is a challenging analytical task due to wild differences in their lipohilicities (hydrophilic decongestants and hydrophobic antihistamines). In this paper a new approach for resolving such a problem is described taking pseudoephedrine sulfate and loratadine as an example. The chromatographic behavior of pseudoephedrine sulfate and loratadine on RP C18 and C8 columns were studied in presence and absence of sodium lauryl sulfate (SLS). The effect of combining two different types of stationary phases (cyano and C18 or C8) on the relative retention of the two compounds was investigated. In conclusion, it was found that the combination of a C18 column followed by a standard cyano column provides a stationary phase that separates both compounds effectively and within a reasonable time. This approach was compared to a literature method and demonstrated to have superior selectivity.     

A new HPLC approach for the determination of hydrophilic and hydrophobic components: the case of pseudoephedrine sulfate and loratadine in tablets

Effective isocratic separations of decongestants and antihistamines is a challenging analytical task due to wild differences in their lipohilicities (hydrophilic decongestants and hydrophobic antihistamines). In this paper a new approach for resolving such a problem is described taking pseudoephedrine sulfate and loratadine as an example. The chromatographic behavior of pseudoephedrine sulfate and loratadine on RP C18 and C8 columns were studied in presence and absence of sodium lauryl sulfate (SLS). The effect of combining two different types of stationary phases (cyano and C18 or C8) on the relative retention of the two compounds was investigated. In conclusion, it was found that the combination of a C18 column followed by a standard cyano column provides a stationary phase that separates both compounds effectively and within a reasonable time. This approach was compared to a literature method and demonstrated to have superior selectivity.     

Capillary-channeled polymer fibers as a stationary phase for desalting of protein solutions for electrospray ionization mass spectrometry analysis

Micropipet solid-phase extraction (SPE) tips have been used to desalt and purify proteins and peptides from mixtures of buffers and biological solutions. Removing salts and buffers prior to electrospray ionization mass spectrometry (ESI-MS) characterization improves the detection limits and the sensitivity of the protein analyses. Recently, capillary-channeled polymer (C-CP) fibers have been investigated as stationary phases for high-performance liquid chromatography separations of proteins. Polypropylene C-CP fibers incorporated as sorbent materials in micro-SPE tips are shown to effectively remove both inorganic and organic buffers from proteins in defined solutions. The architecture of the fibers provides large surface areas in comparison to conventional round fibers and is readily packed into capillaries that can be affixed to micropipet tips. Desalting of protein solutions is demonstrated for ESI-MS analysis through increased signal-to-noise ratios and reduced spectral complexity.     

Gradient chromatofocusing. versatile pH gradient separation of proteins in ion-exchange HPLC: characterization studies

A new chromatofocusing technique called gradient chromatofocusing is characterized. Gradient chromatofocusing generates linear pH gradients on anion-exchange columns with inexpensive low molecular mass buffer components via HPLC gradient mixing. Gradient chromatofocusing results are compared with that of conventional chromatofocusing in the chromatography of several proteins on a Mono P column, including beta-lactoglobulin A and B, ovalbumin, BSA, and conalbumin. Gradient chromatofocusing shows superior performance, with resolution increases greater than 3-fold being realized for the entire protein mixture and up to 25-fold for a particular protein pair. This performance superiority arises from inherent advantages in the gradient chromatofocusing technique in optimizing conditions pertinent to separation, including buffer concentration and pH gradient slope. These resolution gains arise from both increases in separation factor and decreases in peak width achieved with the pH gradient chromatofocusing technique through the manipulation of buffer concentration and the pH gradient profile. Gradient chromatofocusing is also compared with conventional NaCl gradient ion-exchange chromatography using the same Mono P column, demonstrating 3-fold resolution gains, resulting from a 3-fold decrease in peak width. The present work demonstrates the significantly improved performance that gradient chromatofocusing has in protein separations compared to other ion-exchange chromatographic techniques. Mechanisms for the various effects are discussed.     

Photoinduced polymerization for entrapping of octadecylsilane microsphere columns for capillary electrochromatography

A novel fritless capillary column for capillary electrochromatography (CEC) has been developed. The ODS microspheres were packed into a capillary and were then immobilized within an organic polymer prepared in situ through a photopolymerization process. The entrapment conditions were investigated to minimize the effect of the polymer matrix on the chromatographic properties of the packing material. The organic polymer matrix in the microsphere-packed column functions to link microspheres at specific sphere-sphere and sphere-capillary contact points. CEC separations of a PAH test mixture using entrapped columns with different UV illumination times were compared in terms of retention factor and separation efficiency. The optimized entrapped column demonstrated better chromatographic performance than similarly packed columns with conventional inlet and outlet frits. The electrochromatographic separations of hormones and peptides were also demonstrated on entrapped ODS columns.     

Separation orthogonality in temperature-programmed comprehensive two-dimensional gas chromatography

In a comprehensive two-dimensional gas chromatograph, a thermal modulator serially couples two columns containing dissimilar stationary phases. The secondary column generates a series of high-speed secondary chromatograms from the sample stream formed by the chromatogram eluting from the primary column. This series of secondary chromatograms forms a two-dimensional gas chromatogram with peaks dispersed over a retention plane rather than along a line. The method is comprehensive because the entire primary column chromatogram is transmitted through the secondary column with fidelity. One might expect that a two-dimensional separation in which both dimensions are basically the same technique, gas chromatography, would be inefficient because the two dimensions would behave similarly, generating peaks whose retentions correlate across dimensions. Applying a temperature program to the two columns, however, can tune the separation to eliminate this inefficiency. The temperature program reduces the retentive power of the secondary column as a function of progress of the primary chromatogram such that the retention mechanism of the primary column is eliminated from the second dimension. Retention of a substance in the second dimension is then determined by the difference in its interaction with the two stationary phases. Retention times in the second dimension then fall within a fixed range, and the whole retention plane is accessible. In a properly tuned comprehensive two-dimensional chromatogram, retention times in the two dimensions are independent of each other, and the two-dimensional chromatogram is orthogonal. Orthogonality is important for two reasons. First, an orthogonal separation efficiently uses the separation space and so has either greater speed or peak capacity than nonorthogonal separations. Second, retention in the two dimensions of an orthogonal chromatogram is determined by two different and independent mechanisms and so provides two independent measures of molecular properties.     

Application of the thermally tuned tandem column concept to the separation of several families of environmental toxicants

Separations of several families of environmental toxicants were optimized by means of the thermally tuned tandem column (T3C) concept. We use a tandem combination of an octadecylsilane (ODS) and a carbon-coated zirconia (C-ZrO2) column; and tune the selectivity by independently adjusting the isothermal temperatures of the two columns. This results in the change in the contribution that each column makes to the overall retention and selectivity. The separation was optimized by locating the optimum pair of column temperatures which give the best separation of the critical solute pair. For both triazine herbicides and carbamate pesticides samples, dramatically different selectivities and different critical pairs were observed for the two types of phases. Although neither individual phase gave adequate separation, the T3C approach provided baseline separations using only four preliminary trial separations. We also showed that, for the triazine samples, the T3C approach gave a better separation than did conventional mobile phase optimization with an ODS column. The combination of superior selectivity of T3C and high flow rate allows the baseline separation of complex mixtures in just a few minutes.     

Capillary array high-performance liquid chromatography of nucleic acids and proteins

An array of monolithic poly(styrene/divinylbenzene) capillaries with individual column thermostats was constructed to demonstrate its utility for the separation of nucleic acids, proteins, and tryptic digests in combination with UV absorbance detection. Because of polymerization-related variation in surface area of monolithic columns, the concentration of acetonitrile required for elution of DNA fragments in denaturing HPLC may vary sufficiently to affect the degree of denaturation. Modulation of column temperature offers a convenient way to harmonize elution profiles among columns. Individual regulation of column temperature also provides the means to determine rapidly in a single parallel run the optimum temperature for resolution of biomolecules. Given the high reproducibility of separations among columns and the ease with which poly(styrene/divinylbenzene)-based stationary phases can be modified to accommodate different modes of chromatography, such arrays will find broad applicability in proteogenomics.     

Sol-gel poly(ethylene glycol) stationary phase for high-resolution capillary gas chromatography

A sol-gel chemistry-based method was developed for the preparation of highly stable capillary gas chromatography (GC) columns with surface-bonded poly(ethylene glycol) (PEG) stationary phase. Through a single-step procedure, it concurrently provided column deactivation, stationary-phase coating, and chemical immobilization of the coated film. Sol-gel reactions were carried out within fused-silica capillaries that were filled with properly designed sol solutions containing two sol-gel precursors, two different triethoxysilyl-derivatized poly(ethylene glycol)s, two sol-gel catalysts, and a deactivation reagent. Hydrolytic polycondensation reactions led to the formation of a sol-gel coating chemically bonded to the inner walls of the capillary. A number of sol-gel coated fused-silica capillary columns were prepared using sol-gel-active PEG derivatives. These columns demonstrated many inherent advantages, the main being the strong anchoring of the coating to the capillary wall resulting from chemical bonding with the silanol groups on the fused-silica capillary inner surface. This chemical bonding yielded strongly immobilized PEG coatings with outstanding thermal stability (up to 320 degrees C). To our knowledge, such a high thermal stability has not been achieved so far on conventionally prepared PEG GC columns. Sol-gel PEG columns provided excellent chromatographic performances: high number of theoretical plates, excellent run-to-run and column-to-column reproducibility, and pronounced selectivity for a wide range of test solutes. Using n-octadecane as a test solute (k = 7.14), an efficiency value of 3200 theoretical plates/m was obtained on a 10 m x 0.25 mm i.d. fused-silica capillary column. Five sol-gel PEG columns provided RSD values of 1.09% for column efficiency (solute, n-octadecane), 1.37% for retention factor (solute, n-octadecane), and 0.9% for separation factor (for solute pair o- and p-xylene). In five replicate measurements using the same column, RSD values of less than 0.50% for the retention time and 1.36% for retention factor (k) were obtained.     

Preparation and evaluation of proline-based chiral columns

Chiral stationary phases made of readily available proline peptides were prepared and evaluated for general chiral separation. With the proper structural elements, these columns demonstrated broad chiral selectivity. Among the 53 analytes tested, a tetraproline column resolved 31. The separations achieved for these analytes are comparable to those achieved on Whelk O2 column, while still inferior to those achieved on Daicel AD-H and OD-H columns. Number of proline units proves important for chiral recognition, because a control column made with a single proline unit is largely ineffective.     

Instrumental requirements for nanoscale liquid chromatography

Nanoscale liquid chromatography (nano-LC), with packed columns of typically 75 μm i.d. × 15 cm length, packed with C18, 5 μm of stationary phase, and optimal flow rates of 180 nL/min, can be considered as a miniaturized version of conventional HPLC. Using the down-scaling factor, which corresponds to the ratio of the column diameter in square, (d(conv)/d(micro))(2), excellent agreement between the theoretically calculated values and the values obtained using the down-scaling factor (～3800) has been observed. This factor was applied to all system components, including flow rate, injection and detection volumes, and connecting capillaries. Down-scaling of a conventional HPLC system to a nano-LC system is easy to realize in practice and involves using a microflow processor for nanoflow delivery (50-500 nL/min), a longitudinal nanoflow cell (≤3 nL), a microinjection valve (≤ 20 nL), low-dispersion connecting tubing, and zero dead volume connections. Excellent retention time reproducibility was measured with RSD values of ±0.1% for isocratic and ±0.2% for gradient elution. Plates counts of more than 100?000/m indicate the excellent performance of the entire nano-LC system. With minimal detectable amounts of proteins in the low femtomole and subfemtomole ranges (e.g., 520 amol for bovine serum albumin), high mass sensitivity was found, making nano-LC attractive for the microcharacterization of valuable and/or minute proteinaceous samples. Coupling nano-LC with concomitant mass spectrometry using nanoscale ion spray or electrospray interfaces looks very promising and is obviously the next step for future work.     

Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column, and nanoelectrospray tip

Current nano-LC/MS systems require the use of an enrichment column, a separation column, a nanospray tip, and the fittings needed to connect these parts together. In this paper, we present a microfabricated approach to nano-LC, which integrates these components on a single LC chip, eliminating the need for conventional LC connections. The chip was fabricated by laminating polyimide films with laser-ablated channels, ports, and frit structures. The enrichment and separation columns were packed using conventional reversed-phase chromatography particles. A face-seal rotary valve provided a means for switching between sample loading and separation configurations with minimum dead and delay volumes while allowing high-pressure operation. The LC chip and valve assembly were mounted within a custom electrospray source on an ion-trap mass spectrometer. The overall system performance was demonstrated through reversed-phase gradient separations of tryptic protein digests at flow rates between 100 and 400 nL/min. Microfluidic integration of the nano-LC components enabled separations with subfemtomole detection sensitivity, minimal carryover, and robust and stable electrospray throughout the LC solvent gradient.     

Comprehensive off-line, two-dimensional liquid chromatography. Application to the separation of peptide digests

The separation of the peptide digests of myoglobin and bovine serum albumin was performed with an off-line combination of two commercial, conventional HPLC columns. The first column was packed with a strong ion exchanger and eluted with a KCl gradient. The second column was packed with particles of C18-bonded silica and eluted with an acetonitrile gradient. The conditional peak capacities of the 2D separations achieved exceed 7000 under the experimental conditions investigated. This performance is achieved at the cost of an analysis time of the order of 28 hours. Possible improvements to the separation method described here are discussed.     

Generating multiple independent retention index data in dual-secondary column comprehensive two-dimensional gas chromatography

A method producing simultaneously three retention indexes for compounds has been developed for comprehensive two-dimensional gas chromatography by using a dual secondary column approach (GC x 2GC). For this purpose, the primary flow of the first dimension column was equally diverted into two secondary microbore columns of identical geometry by means of a three-way flow splitter positioned after the longitudinally modulated cryogenic system. This configuration produced a pair of comprehensive two-dimensional chromatograms and generated retention data on three different stationary phases in a single run. First dimension retention indexes were determined on a polar SolGel-Wax column under linear programmed-temperature conditions according to the van den Dool approach using primary alcohol homologues as the reference scale. Calculation of pseudoisothermal retention indexes in both second dimensions was performed on low-polarity 5% phenyl equivalent polysilphenylene/siloxane (BPX5) and 14% cyanopropylphenyl/86% dimethylpolysiloxane (BP10) columns. To construct a retention correlation map in the second dimension separation space upon which KovAts indexes can be derived, two methods exploiting "isovolatility" relationships of alkanes were developed. The first involved 15 sequential headspace samplings of selected n-alkanes by solid-phase microextraction (SPME), with each sampling followed by their injection into the GC at predetermined times during the chromatographic run. The second method extended the second dimension retention map and consisted of repetitive introduction of SPME-sampled alkane mixtures at various isothermal conditions incremented over the temperature program range. Calculated second dimension retention indexes were compared with experimental values obtained in conventional one-dimensional GC. A case study mixture including 24 suspected allergens (i.e., fragrance ingredients) was used to demonstrate the feasibility and potential of retention index information in comprehensive 2D-GC.     

Fabrication of graphene oxide nanosheets incorporated monolithic column via one-step room temperature polymerization for capillary electrochromatography

Graphene oxide (GO) has received great interest for its unique properties and potential diverse applications. Here, we show the fabrication of GO nanosheets incorporated monolithic column via one-step room temperature polymerization for capillary electrochromatography (CEC). GO is attractive as the stationary phase for CEC because it provides not only ionized oxygen-containing functional groups to modify electroendoosmotic flow (EOF) but also aromatic macromolecule to give hydrophobicity and π-π electrostatic stacking property. Incorporation of GO into monolithic column greatly increased the interactions between the tested neutral analytes (alkyl benzenes and polycyclic aromatics) and the stationary phase and significantly improved their CEC separation. Baseline separation of the tested neutral analytes on the GO incorporated monolithic column was achieved on the basis of typical reversed-phase separation mechanism. The precision (relative standard deviation (RSD), n = 3) of EOF was 0.3%, while the precision of retention time, peak area, and peak height for the tested neutral analytes were in the range of 0.4-3.0%, 0.8-4.0%, and 0.8-4.9%, respectively. In addition, a set of anilines were well separated on the GO incorporated monolith. The GO incorporated monolithic columns are promising for CEC separation.     

Selective retention of oxygen using chromatographic columns containing metal chelate polymers

Porous polymers containing various metal chelates bonded to nitrogen functionalities on the surface of the polymer have been synthesized and found to bind oxygen reversibly. The stationary phases containing [5,5'-(1,2-ethanediyldinitrilo)-bis(2,2,7-trimethyl-3-octanonato)]cobalt(II) were found to be the most suitable of the phases investigated for separating oxygen for argon, nitrogen, and carbon monoxide. At ambient temperatures, near 25 degrees C, the reversible interaction of molecular oxygen with the transition-metal complex bonded to the stationary phase results in a marked increase in the retention time of oxygen, relative to species that have similar retention times in columns that do not contain the metal chelate. The stationary phase can be used alone to achieve the separation of low molecular weight gases or in series with another column. The metal chelate stationary phase is selective for oxygen and little change in the retention time of oxygen is observed after hundreds of injections over a several-month period, indicating that no appreciable degradation of the stationary phase had taken place under these conditions.     

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.     

Microchip HPLC of peptides and proteins

Rapid microchip reversed-phase HPLC of peptides and proteins at pressure gradients of 12 bar/cm (180 psi/cm) has been performed using a microdevice that integrates subnanoliter on-chip injection and separation with a miniaturized fluorescence detector. Proteins and peptides were separated on a C18 side-chain porous polymer monolith defined by contact lithography, and injection was achieved via a pressure-switchable fluoropolymer valve defined using projection lithography. Preliminary separations of peptide standards and protein mixtures were performed in 40-200 s, and switching between samples with no detectible sample carryover has been performed. The injections and separations were reproducible; the relative standard deviation (RSD) for retention time was 0.03%, and peak area RSD was 3.8%. Sample volumes ranging from 220 to 800 pL could be linearly metered by controlling the pressure injection pulse duration with conventional timing and valving. The current prototype system shows the potential for rapid and autonomous HPLC separations with varying modalities and the potential for direct connection to mass spectrometers at nanospray flow rates.