Fundamental studies of liquid chromatography at the critical condition using enhanced-fluidity liquids

The improvement in the analysis of telechelic polymer matrixes continues to be a pursuit for many scientists of varying disciplines. This quest for a new technique has led to the continued development of liquid chromatography at the critical condition (LCCC) or liquid chromatography at the critical adsorption point (LC-CAP). LCCC allows for the isolation of one area of the polymer matrix so that other areas of the polymer can be probed with size-exclusion or adsorptive chromatographic modes. Although this technique has been successfully applied to the analysis of telechelic polymers, the practice of LCCC can be difficult. These difficulties include finding and maintaining a solvent system appropriate for the practice of LCCC as well as deterioration of peak shape once the system is operating at the LCCC mode. Because of the specificity of the mobile phase required for the practice of LCCC, the work is routinely practiced by premixing solvents. Previous work with enhanced-fluidity liquid mobile phases demonstrated that these mobile phases removed many of the aforementioned challenges associated with working at the LCCC mode. These mobile phases utilize both pressure and temperature variation in order to maintain the specific solvent strength necessary for the LCCC work. This work studies the coupling and optimization of enhanced-fluidity, EF, liquid mobile phases for LCCC. Several EF-LCCC systems, differing in mobile phase composition, temperature, and pressure, were routinely established, resulting in the effective practice of critical chromatography. The practice of LCCC with on-line mobile phase preparation is demonstrated using commercially available instrumentation. Finally, EF-LCCC is used to analyze triblock and diblock copolymers.     

Initial characterization of humic acids using liquid chromatography at the critical condition followed by size-exclusion chromatography and electrospray ionization mass spectrometry

Structural information on humic acids is difficult to obtain because of the heterogeneity of the acids. Herein liquid chromatography at the critical condition, LCCC, is used to provide a sorting mechanism for the diverse types of molecules contained in humic acids. The critical condition of polymers that are believed to model some subunit of the humic acid is determined. Humic acids from three different terrestrial sources (soil, compost, and peat) are then separated under these chromatographic conditions. The portion of the humic acid that has structure similar to that of the model polymer elutes at the retention volume of the critical condition of the model. Next, fractions are collected and further characterized. This detailed characterization includes high-efficiency size-exclusion chromatography and electrospray mass spectrometry. The size-exclusion chromatograms of the fractions were found to be markedly different from that of the original humic acid sample. This is strong evidence that the LCCC separation mechanism is different from size fractionation. The mass spectra of the humic acid fractions were also markedly different from those of the bulk humic acids previously reported. The mass spectra of specific fractions collected had repeating clusters of m/z values, which is more evidence that the critical condition separation is a powerful sort function.     

In-depth characterization of slurry packed capillary columns with 1.0-microm nonporous particles using reversed-phase isocratic ultrahigh-pressure liquid chromatography

Fused-silica capillary columns packed with 1.0-microm nonporous C18 bonded particles are evaluated with isocratic ultrahigh-pressure liquid chromatography (UHPLC). Improved UHPLC techniques have demonstrated column efficiencies as high as 730 000 plates/m and run pressures over 6800 bar (100 000 psi) for packed 10-microm-inner diameter (i.d.) columns. Columns as large as 150 microm have been tested with UHPLC and show no flow-induced heating effects on separation efficiencies. van Deemter plot analysis for column i.d.s ranging from 10 to 150 microm shows an increase in column efficiency with a decrease in column i.d. Reduced parameter analysis further illustrates a decrease in reduced parameter A term and C term values with decreasing i.d. However, reduced parameter C term values for columns evaluated with UHPLC are an order of magnitude larger than C term values for larger particles at conventional pressures. Retention factors for moderately retained compounds are observed to increase with column i.d., suggesting an increase in packing density. Highly ordered packing arrangement at the column wall is seen for packed beds extruded from large-diameter columns.     

Development of water-based liquid chromatography at the critical condition

Liquid chromatography at the critical condition (LCCC) is a chromatographic technique that allows for the isolation of one area of the polymer matrix so that other areas of the polymer may be probed with size-exclusion or adsorptive chromatographic modes. This technique has been successfully applied to the analysis of functionality distributions in functionalized oligomers and to polymer distributions within copolymers. Herein, the critical conditions of two polar polymers, poly(acrylic acid) and polystyrene sulfonate, are determined. These conditions were identified by varying buffer concentration, organic modifier within the mobile phase, or both. At the critical condition of poly(acrylic acid), the retention characteristics of a copolymer of acrylic acid and vinyl pyrrolidinone were determined. This extension to water-based mobile phase conditions will substantially broaden the possible applications of LCCC.     

Synthesis and characterization of capillary columns coated with glycoside-bearing polymer

Fused silica capillary columns were coated with a polymer obtained by radical polymerization of a novel acrylamido derivative, [(N-acryloylamino)ethoxy]ethyl-β-d-glucopyranose (AEG). This monomer, bearing a monosaccharide residue, was synthesized by direct coupling of [(N-acryloylamino)ethoxy]ethanol with glucose. The aim of this work was to demonstrate that weak, reversible interactions that take place between wall polymer coatings and protein analytes lead to poor reproducibility of transit times and capillary performance degradation. The pronounced hydrophilicity of the new poly(AEG) phase improves the reproducibility of transit times and the peak efficiency, as demonstrated by a complete series of tests carried out at various pH values.     

Liquid chromatography at the critical condition for polyisoprene using a single solvent

Liquid chromatography at the chromatographic critical condition has drawn much attention as an attractive characterization method of block copolymers since it has been proposed that a part of a polymer chain becomes "chromatographically invisible" at this condition, which would permit the characterization of individual blocks. A critical condition for a polymer species has been commonly established by use of mixed-solvent systems. It is not easy, however, to reproduce the critical condition since the retention of polymers depends very sensitively on the solvent composition and purity. Furthermore, the preferential sorption of a component in a mixed solvent may cause an additional problem. Therefore, the use of a single solvent is highly desirable to improve the reproducibility as well as the repeatability. In this study, a single-solvent critical condition for polyisoprene was established with 1,4-dioxane and C18 bonded silica as the mobile and stationary phases, respectively. At this condition, the "chromatographic invisibility" of polystyrene-polyisoprene diblock copolymers was critically examined and it was found that a rigorous chromatographic invisibility was not achieved and the retention of the block copolymers was affected by the length of the blocks under the critical condition. Some other chromatographic applications using the single-solvent system are also reported.     

Detection of peptides by precolumn derivatization with biuret reagent and preconcentration on capillary liquid chromatography columns with electrochemical detection

The separation and detection of biuret complexes of neuropeptides by capillary liquid chromatography with electrochemical detection was explored. Capillaries of 25-micron inner diameter packed with base-resistant, polymer-based reversed-phase particles were used for separation, and C-fiber electrodes were used for detection. Detection at the C-fiber electrode was found to have some differences in relative sensitivity for peptides compared to glassy carbon electrodes used previously. On-column preconcentration of preformed complexes allowed up to 1-microL samples to be injected with minimal band broadening resulting in a 100-fold improvement in concentration detection limit with no effect on mass detection limit. Concentration detection limits ranged from 5 to 59 pM, depending upon the peptide, corresponding to 5-59 amol injected. The low concentration detection limit was possible because of minimal baseline disturbances, minimal formation of unwanted products, and high efficiency of complex formation associated with biuret derivatization. The method was applied to determination of vasopressin and bradykinin in dialysates collected with 5-min sampling frequency from the rat supraoptic nucleus.     

Protein-doped monolithic silica columns for capillary liquid chromatography prepared by the sol-gel method: applications to frontal affinity chromatography

The development of bioaffinity chromatography columns that are based on the entrapment of biomolecules within the pores of sol-gel-derived monolithic silica is reported. Monolithic nanoflow columns are formed by mixing the protein-compatible silica precursor diglycerylsilane with a buffered aqueous solution containing poly(ethylene oxide) (PEO, MW 10,000) and the protein of interest and then loading this mixture into a fused-silica capillary (150-250-microm i.d.). Spinodal decomposition of the PEO-doped sol into two distinct phases prior to the gelation of the silica results in a bimodal pore distribution that produces large macropores (>0.1 microm), to allow good flow of eluent with minimal back pressure, and mesopores (approximately 3-5-nm diameter) that retain a significant fraction of the entrapped protein. Addition of low levels of (3-aminopropyl)triethoxysilane is shown to minimize nonselective interactions of analytes with the column material, resulting in a column that is able to retain small molecules by virtue of their interaction with the entrapped biomolecules. Such columns are shown to be suitable for pressure-driven liquid chromatography and can be operated at relatively high flow rates (up to 500 microL x min(-1)) or with low back pressures (<100 psi) when used at flow rates of 5-10 microL x min(-1). The clinically relevant enzyme dihydrofolate reductase was entrapped within the bioaffinity columns and was used to screen mixtures of small molecules using frontal affinity chromatography with mass spectrometric detection. Inhibitors present in compound mixtures were retained via bioaffinity interactions, with the retention time being dependent on both the ligand concentration and the affinity of the ligand for the protein. The results suggest that such columns may find use in high-throughput screening of compound mixtures.     

Separation and detection of phosphorylated and nonphosphorylated peptides in liquid chromatography-mass spectrometry using monolithic columns and acidic or alkaline mobile phases

Efficient chromatographic separation is a prerequisite for the sensitive analysis of complex peptide mixtures using liquid chromatography-mass spectrometry. This is especially true for the analysis of mixtures of unmodified and posttranslationally modified peptides, for example, phosphorylated peptides in the presence of their unmodified analogues. Applying monolithic capillary columns based on poly(styrene/divinylbenzene), the influence of acidic eluents based on trifluoroacetic and heptafluorobutyric acid as well as an alkaline eluent based on triethylamine-acetic acid (pH 9.2) on the separation of synthetic phosphopeptides was evaluated. Heptafluorobutyric acid offered the longest retention times and highest selectivities and, hence, the most effective separation. Application of the alkaline eluent in conjunction with detection in negative ion mode electrospray ionization mass spectrometry, on the other hand, allowed the detection of phosphorylated peptides with significantly lower limits of detection, as compared to acidic eluents in combination with detection in positive ion mode. Pairs of phosphorylated and nonphosphorylated synthetic peptides, ranging from 7- to 16-mers, as well as phosphorylated peptides form a tryptic protein digest could be separated both at acidic and alkaline pH. Utilizing a 60 x 0.20-mm-i.d. capillary column, the limit of detection in negative ion detection mode for a 4-fold phosphorylated peptide in a beta-casein digest was 10 fmol. Together with the capability for fast separation of protein digests, monolithic columns, thus, facilitate the effective and sensitive analysis of this important posttranslational modification.     

Protein characterization by on-line capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry

Two-dimensional polyacrylmide gel electrophoresis (2D-PAGE), perhaps the most widely used method in proteomics research, is often limited by sensitivity and throughput. Capillary isoelectric focusing (CIEF) coupled with electrospray ionization (ESI) mass spectrometry (MS) provides a liquid-based alternative to 2D-PAGE that can overcome these problems but is limited by ampholyte interference and signal quenching in ESI-MS. Inserting a reversed-phase liquid chromatography (RPLC) step between CIEF and MS can remove this interference. In this work, a CIEF-RPLC-MS system is described for separation and characterization of proteins in complex mixtures. CIEF is performed with a microdialysis membrane-based cathodic cell that also permits protein fractions to be collected, washed to remove ampholyte, and analyzed by RPLC-MS. CIEF performance with this cell is equivalent to that achieved with a conventional cathodic cell, and no loss of protein is observed during faction collection. The cell can be easily and safely retrofitted into commercial instrumentation and is applicable for peptide analysis as well. Protein detection at the low-femtomole level is demonstrated with little or no interference from ampholyte, and CIEF-RPLC-MS data are used to construct a plot of pI vs MW for a protein mixture. The current instrumental configuration allows seven fractions in the pI range 3-10 to be analyzed by RPLC-MS in 2 h.     

Combination of column temperature gradient and mobile phase flow gradient in microcolumn and capillary column high-performance liquid chromatography

The combination of several gradient modes (solvent, temperature, and flow programming) is rarely used in HPLC analysis. In this work, the separations obtained utilizing simultaneous flow and temperature gradient in capillary column and microcolumn HPLC were compared with the separations performed under isocratic, isothermal, and isorheic (constant flow) conditions. When the mobile phase flow rate and the column temperature were changed simultaneously during the separation run, the analysis time was shortened up to 50%, while the separation efficiency was preserved. The separations obtained with combined temperature and flow gradients show high reproducibility (relative standard deviation <2.0%), comparable to the reproducibility normally seen with a mobile phase gradient. For capillary HPLC, simultaneous temperature and flow programming is the method of choice because of the great technical difficulties involved in performing solvent gradient elution.     

Determination of acaricides in honey by liquid chromatography with ordinary, narrow-bore, and microbore columns

Narrow-bore and microbore columns packed with octadecylsilane were used to compare their sensitivity and efficiency in the separation of coumaphos, fluvalinate, bromopropylate, and 4,4'-dibromobenzophenone from honey with those of ordinary columns. The best sensitivity for acaricides was accomplished by using a 150 mm × 0.32 mm i.d., 5 μm Spherisorb ODS-2 capillary column, methanol-water (90:10 v/v) as the mobile phase, and 5 μL/min as the flow rate. Detection limits for individual acaricides using a UV detection range from 0.40 to 0.74 μg/kg of honey were comparable to those obtained by gas chromatography using an electron capture detector. All acaricides were separated in <12 min. The coefficients of variations on real samples were <6.2%.     

Mass spectrometric characterization of functional poly(methyl methacrylate) in combination with critical liquid chromatography

State-of-the-art techniques for the mass spectrometric characterization of synthetic polymers have been applied to functional poly(methyl methacrylate), synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization. The polymers were first separated effectively according to functionality by liquid chromatography (LC) at the critical conditions (i.e., almost no influence of molecular weight on retention). The separated polymers were characterized off-line by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and both off-line and on-line by LC-electrospray-ionization-quadrupole-TOF-MS (LC-ESI-QTOF- MS). The on-line ESI experiments confirmed a clear baseline separation of the hydroxyl-functional prepolymers according to the number of hydroxyl groups. Labile end groups of PMMA, such as the dithioester group, were lost in the MALDI-TOF-MS experiments, while they were observed intact in the ESI-QTOF-MS spectra. This indicates that in the present case ESI is a much softer ionization technique than is MALDI. The ESI-MS experiments provided direct evidence that the RAFT polymers still exhibited living characteristics in the form of the dithio moiety.     

Fritless capillary columns for HPLC and CEC prepared by immobilizing the stationary phase in an organic polymer matrix

A new design of immobilized particle separation media for capillary liquid chromatography and electrochromatography has been developed. A mixture of porogenic solvents and methacrylate-based monomers is pumped through a packed column to provide, following a polymerization step, an organic matrix capable of holding the sorbent particles in place, thus rendering the end frits unnecessary. The new columns demonstrate excellent chromatographic performance in both CEC (reduced plate height [h]=1.1-1.5) and micro LC modes (h = 2.2-2.5), while minimizing secondary interactions encountered when silica-based entrapment matrixes are employed. In addition to delivering mechanically robust columns, the methacrylate matrix provides a mechanism for fine tuning of the electroosmotic flow velocity when 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) is incorporated into the polymerization mixture.     

Detection of in vivo formed DNA adducts at the part-per-billion level by capillary liquid chromatography/microelectrospray mass spectrometry

Capillary liquid chromatography/microelectrospray mass spectrometry has been applied to the detection of deoxyribonucleoside adducts of the food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) from in vivo sources. Adjustments were made to a previously described methodology such that analyte detection could be improved by nearly 2 orders of magnitude. These adjustments included changing the electrospray ionization sprayer configuration, increasing the sample injection volume, improving the solid-phase extraction procedure, and increasing peak efficiency by modifying chromatographic conditions. While this scheme for improving analyte detection was targeted for DNA adducts, it could be applied to almost any LC/MS methodology where sensitive analysis is the primary objective. Selective reaction monitoring) techniques with a triple quadrupole mass spectrometer enabled sensitive and specific detection of IQ adducts, with detection limits approaching 1 adduct in 10(9) unmodified bases using approximately 500 microg of DNA. The DNA adducts N-(2'-deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline and 5-(2'-deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline were detected in pancreas tissue of a cynomolgus monkey sacrificed 24 h after a single administration of 10 mg/kg carcinogen. The LC/MS results were consistent with previously published 32P-postlabeling data (Turesky et al. Chem Res. Toxicol. 1996, 9, 403-408). Thus, capillary tandem LC/MS is a highly sensitive technique, which can be used to screen for DNA adducts in vivo.     

Characterization of poly(L-lactide)-block-poly(ethylene oxide)-block-poly(L-lactide) triblock copolymer by liquid chromatography at the critical condition and by MALDI-TOF mass spectrometry

Poly(L-lactide)-block-poly(ethylene oxide)-block-poly(L-lactide) triblock copolymers (PLLA-b-PEO-b-PLLA) were fractionated in terms of the number of LLA units by liquid chromatography at the critical condition (LCCC) of PEO block. The fractionated samples were identified using MALDI-TOF mass spectrometry. The dependence of the LCCC retention of the diblock and triblock copolymers on the degree of polymerization of PLLA block(s) follows Martin's rule very well. Unlike the case of PEO-b-PLLA diblock copolymer reported earlier (Lee, H.; et al. Macromolecules 1999, 32, 4143), however, a splitting of the elution peaks containing the same number of LLA units was found. The peak splitting was ascribed to the different length distributions of PLLA blocks at the two ends of the PEO block. From the relative intensities of the peaks, the split peaks were assigned to different isomeric structures of the PLLA blocks. From these results we conclude that the interaction of the triblock copolymers with the stationary phase is affected by the distribution of the interacting blocks at the two ends of the center PEO block, in addition to the total number of LLA units in the triblock copolymer.     

High-sensitivity peptide mapping by capillary zone electrophoresis and microcolumn liquid chromatography, using immobilized trypsin for protein digestion

Procedures for the reduced-scale analysis of proteins by peptide mapping have been developed, allowing peptide maps to be obtained from picomole to femtomole quantities of protein. The use of trypsin immobilized on agarose gel and placed in a small reactor column has made it possible to reproducibility digest as little as 50 ng of protein. This represents a decrease in sample size of approximately 3 orders of magnitude from conventional tryptic digestion schemes. Separations of tryptic digests were accomplished by using either microcolumn high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). Separations of 100 ng (4 pmol) of tryptic digest samples of beta-casein were achieved with microcolumn HPLC, while separations of approximately 2 ng (80 fmol) of beta-casein tryptic digest (from a total sample size of 50 ng) were possible with CZE. Peptide maps from phosphorylated and dephosphorylated forms of beta-casein were readily distinguishable using both separation methods, demonstrating an ability to detect a single amino acid modification in a protein. Relative standard deviations of peak retention or migration times were less than 3% for microcolumn HPLC and less than 1% for CZE.     

Dynamics of single-protein molecules at a liquid/solid interface: implications in capillary electrophoresis and chromatography

The behavior of individual molecules of R-phycoerythrin (RPE) was monitored by fluorescence imaging at various pHs and ionic strengths within the evanescent-field layer (EFL) at a water/fused-silica interface. Above the isoelectric point (pI), the individual protein molecules moved between exposures with random motion. As the pH approached the pI of the protein, the RPE molecules were partially adsorbed onto the fused-silica surface. The residence time and the number of molecules within the EFL also increased near the pI. Below the pI, the protein molecules were completely and permanently adsorbed onto the surface. However, the observed number of distinct molecule spots was decreased somewhat because of aggregation. At a given buffer condition, plots of residence times and molecule numbers exhibit asymmetry nearly identical to the corresponding elution peaks of the proteins in capillary electrophoresis and capillary liquid chromatography. These results provide insights into the fundamental interactions for the adsorption/desorption of proteins at the liquid/solid interface.