High‐resolution peptide separations using nano‐LC at ultra‐high pressure

We report on the optimization of nano‐LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 μm id capillary columns packed with 2, 3, and 5 μm fully porous silica C18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 μm particle‐packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic‐performance limit, i.e. using 2‐μm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run‐to‐run retention time stability (RSD values below 0.18%) was demonstrated applying ultra‐high pressure conditions.     

Solvent gradient elution in micro HPLC

A simple gradient method for capillary column and micro high performance liquid chromatography using either single or multiple mixing chambers is presented. The gradient profile is dependent on the number of the mixing chambers. This system permits solvent gradient elution at flow rates less than 10 μl/min with satisfactory reproducibility.     

A miniature gradient elution system for liquid chromatography with packed capillary columns

A miniaturized continuous gradient elution system was designed for work with packed capillary columns. The retention reproducibility achieved is adequate for many practical applications.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

Coupled LC-GC: Evaporation rates for partially concurrent eluent evaporation using an early solvent vapor exit

Partially concurrent eluent evaporation presupposes an eluent evaporation rate in the GC pre-column that approaches the LC flow rate. Discharging the vapors through the whole GC column, evaporation rates reach 10–30 μl/min, i.e. are suitable just for LC flow rates typical for packed capillary LC columns. With an early vapor exit, evaporation rates are increased to 100–200 μl/min (under extreme conditions to some 800 μl/min), thus fitting the LC flow rates of 2 mm i.d. columns. Evaporation rates were measured for a standard set of pre-columns and conditions. The dependence of the evaporation rate on temperature, inlet pressure, carrier gas, and internal diameter of the retaining pre-column are discussed in order to allow the design of a GC system producing a desired evaporation rate.     

An electroosmotic pump for packed capillary liquid chromatography

An electroosmotic pump (EOP) capable of generating pressure above 3 MPa and μl/min flow rate with reverse phase mobile phases of HPLC was constructed and evaluated. The pump consisted of three parallel connected fused silica capillary columns (25 cm×320 μm I.D.) packed with 2 μm silica materials, hollow electrodes, a high voltage DC power supply, and a liquid pressure transducer. The EOP was applied in a capillary liquid chromatographic system for mobile phase delivery instead of a mechanical pump. Standard samples containing thiourea, naphthalene, anthracene, phenanthrene and acetonitrile were separated on a 15 cm×320 μm I.D. 5 μm Chromasil C₁₈ packed capillary column with acetonitrile/water as mobile phase.     

Application of capillary reversed-phase high-performance liquid chromatography to high-sensitivity protein sequence analysis.

A continuous gradient elution method for capillary column (less than 0.32 mm I.D.) liquid chromatography was developed. Gradient eluent from a microbore liquid chromatograph was split ahead of the injector so that an accurate percentage (2-3%) of the mobile phase delivered by the pump flowed through the capillary column. The outlet of the column was connected to a length of 0.075 mm I.D. fused-silica capillary tubing which, in turn, was connected to a 6-mm optical path length longitudinal capillary flow cell. Fused-silica capillary columns of 0.32 mm I.D. were slurry-packed efficiently with 7-microns spherical, 300 A pore size, C8 bonded-phase particles, and evaluated in terms of their ability to resolve mixtures of proteins, peptides or phenylthiohydantoin (PTH)-amino acid derivatives. The gradient elution profiles agreed with those obtained using microbore (less than 2.1 mm I.D.) and larger bore columns. The minimum detectable amounts for proteins and PTH-amino acids on 0.32 mm I.D. capillary columns were 50 pg and 25 fmol, respectively. At a flow-rate of 3.6 microliters/min, proteins and peptides were recovered from the capillary columns in volumes of about 2-8 microliters. The use of a multiple-wavelength, forward-optics detector for identifying tryptophan- and tyrosine-containing peptides is discussed.     

High resolution liquid chromatography with a packed micro glass capillary column

Single, long columns (1–5 m) can be prepared efficiently using reversed phase packings (3–10 μm particle diameter). 1-m columns packed with 3 and 10 μm packing provide 110 000 and 50 000 theoretical plates, respectively. Very efficient columns can resolve highly complex mixtures and difficult-to-separate compounds. Temperature gradient elution is a powerful technique for LC with microbore columns.     

Characterization and kinetic performance of 2.1 × 100 mm production columns packed with new 1.6 μm superficially porous particles

The overall kinetic performance of three production columns (2.1 mm × 100 mm format) packed with 1.6 μm superficially porous CORTECS‐C₁₈+ particles was assessed on a low‐dispersive I‐class ACQUITY instrument. The values of their minimum intrinsic reduced plate heights (h_min = 1.42, 1.57, and 1.75) were measured at room temperature (295 K) for a small molecule (naphthalene) with an acetonitrile/water eluent mixture (75:25, v/v). These narrow‐bore columns provide an average intrinsic efficiency of 395 000 plates per meter. The gradient separation of 14 small molecules shows that these columns have a peak capacity about 25% larger than similar ones packed with fully porous BEH‐C₁₈ particles (1.7 μm) or shorter (50 mm) columns packed with smaller core–shell particles (1.3 μm) operated under very high pressure (>1000 bar) for steep gradient elution (analysis time 80 s). In contrast, because their permeabilities are lower than those of columns packed with larger core–shell particles, their peak capacities are 25% smaller than those of narrow‐bore columns packed with standard 2.7 μm core–shell particles.     

Tryptic digest mapping by gradient capillary electrochromatography

A high performance liquid chromatography (HPLC) system complemented with T-split, capillary detection cell and a high voltage power supply was used for peptide mapping by gradient electrochromatography and nanoliquid chromatography (nano-LC). With capillary columns of 100 microm ID, 6 cm packed with octadecylated 1.5 microm silica particles, the typical analysis time was approximately 10-15 min. The resolution of a tryptic digest of cytochrome c obtained by electrochromatography at 100 kV/m was superior compared to the analysis by nano-LC. Bubble formation caused by Joule heating at currents up to 100 microA was successfully suppressed by using a resistor capillary of 25 microm ID connected to the outlet of the packed column.     

An initial assessment of the use of gradient elution in microemulsion and micellar liquid chromatography

Novel microemulsion and micellar HPLC separations have been achieved using gradient elution and columns packed with reverse phase material. Initial attempts at gradient microemulsion liquid chromatography proved impossible on use of a microemulsion successfully used in capillary electrophoresis. Optimisation of the microemulsion composition allowed the generation of stable microemulsions to achieve separations in HPLC. The novel use of organic-solvent micellar chromatography in gradient elution mode was shown to give efficient separations. A range of efficient separations of pharmaceuticals and related impurities were obtained. Acidic, basic, and neutral solutes were resolved covering a wide range of water solubilities and polarities. Elution times were in the order of 4-15 minutes. Separations were briefly compared to those accomplished with a micellar HPLC system. It is proposed that gradient elution in both microemulsion and micellar HPLC can be regarded as a highly successful means of achieving resolution of complex mixtures and should be considered for routine analysis and further investigation.     

Silica-based monoliths for rapid peptide screening by capillary liquid chromatography hyphenated with electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Capillary liquid chromatography based on particulate and monolithic stationary phases was used to screen complex peptide libraries by fast gradient elution coupled on-line to electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS). A slightly modified commercial electrospray interface consisting of a fused-silica transfer capillary and low dead volume stainless steel union at which the electrospray voltage was grounded enabled the effluent of all the capillary columns to be directly sprayed into the mass spectrometer. Stable electrospray conditions were generated over a wide range of mobile phase compositions, alleviating the need for a tapered end of the spray capillary, pneumatic assistance or preheated nebulizer gas. Since the identification of complex samples containing numerous isobaric substances is facilitated by chromatographic separation prior to mass spectrometry, stationary phase materials have been employed which offer a fast, efficient elution and, due to the complexity of samples, a high loading capacity. Silica-based monolithic capillary columns combine these three characteristics in a unique manner due to a tailored adjustment of both macro- and mesopore sizes in the highly porous silica structure. As we demonstrate by a comparative study of the silica-based monolithic and packed capillaries for LC/MS analysis of complex peptide libraries, silica monoliths show superior performance over packed beds of small-diameter particles with respect to analysis time and separation efficiency. Libraries with more than 1000 different peptides could be screened in less than 20 min.     

Polymetacrylate and hybrid interparticle monolithic columns for fast separations of proteins by capillary liquid chromatography

Preparation of organic polymer monolithic columns in fused silica capillaries was aimed at fast gradient separation of proteins. For this purpose, polymerization in situ procedure was optimized, using ethylene dimetacrylate and butyl metacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in presence of non-aqueous porogen solvent mixtures composed of 1-propanol and 1,4-butanediol. The separation of proteins in totally monolithic capillary columns was compared with the chromatography on a new type of "hybrid interparticle monolithic" capillary columns, prepared by in situ polymerization in capillary packed with superficially porous spherical beds, 37-50 microm. The "hybrid" columns showed excellent stability and improved hydrodynamic flow properties with respect to the "totally" monolithic capillary columns. The separation selectivity is similar in the two types of columns. The nature of the superficially porous layer (bare silica or bonded C18 ligands) affects the separation selectivity less significantly than the porosity (density) of the monolithic moiety in the interparticle space, controlled by the composition of the polymerization mixture. The retention behaviour of proteins on all prepared columns is consistent with the reversed-phase gradient elution theory.     

Analysis of ppb levels of organics in water by means of purge-and-trap, capillary gas chromatography and selective detectors

The Environmental Protection Agency (EPA) has issued a series of methods (500 and 600 series) for the analysis of organics in drinking water and industrial discharges. Methods 601 and 602 employ packed-column gas chromatography (GC) with electrolytic conductivity (E1CD) and photoionization detection (PID), respectively. A purge-and-trap system is used for concentration of volatiles. The EPA is in the process of converting methods 601 and 602 and certain 500-series methods to capillary column analysis. We have also initiated the conversion of a number of these methods, using E1CD and PID and have described them in detail in this paper. We have evaluated both 0.32- and 0.53-mm diameter capillary columns, using helium and nitrogen as carrier gases with each detector. We found that with nitrogen optimum results are obtained at a flow-rate of 15 ml/min, and with helium at 6-8 ml/min. As a result of system optimization, including operation of the two detectors in series, and converting from packed to capillary columns, we found that analysis time could be reduced from 80 min (for two methods separately) to ca. 30 min. In addition, the elution of more than five components in one peak, observed when the packed column specified in method 601 are used, was eliminated.     

Effects of column length, particle size, gradient length and flow rate on peak capacity of nano-scale liquid chromatography for peptide separations

The effects of the column length, the particle size, the gradient length and the flow rate of a nanoLC system on peptide peak capacity were investigated and compared. Columns packed with 1.7 microm and 3 microm C(18) materials into pieces of 75 microm capillary tubing of various lengths were tested with different gradient lengths and flow rates. While increasing the length of a column packed with the 1.7 microm material helped improve peptide peak capacity at the whole range of the tested gradient lengths (24-432 min), little improvement in peak capacity was observed with the increase of the length of a column packed with the 3 microm material unless a gradient longer than 50 min was carried out. Up to 30% of peak capacity increase was observed when a column's length is doubled, with little reduction in the throughput. In most cases, more than 50% of the increase in peak capacity was obtained with the reduction in the particle size from 3 microm to 1.7 microm. With the same backpressure generated, a shorter 1.7-microm-particle column outperformed a longer column packed with the 3 microm material. In a flow rate range of 100-700 nl/min, increasing the flow rate improved peak capacity for columns packed with 1.7 microm and 3 microm materials.     

Preparation of monodisperse porous polymethacrylate microspheres and their application in the capillary electrochromatography of macrolide antibiotics

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were prepared by a simple one-step dispersion polymerization process. Examination of the polymeric microspheres showed that they had a mean particle diameter of 3 microm and dual pore size distribution with mean pore diameters of 300 and 800 A. The microspheres were functionalized by introducing quaternary ammonium/octadecyl groups to obtain positively charged beads in a wide pH range. The functionalized beads were packed into fused-silica capillary having 50 microm inner diameter and used to separate erythromycin derivatives by capillary electrochromatography (CEC). These samples require gradient elution when separated by high-performance liquid chromatography (HPLC) or micro-HPLC, but with the new columns isocratic elution suffices for their separation by CEC. The column efficiency ranged from 40,000 to 50,000 theoretical plates.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

Highly efficient monolithic silica capillary columns modified with poly(acrylic acid) for hydrophilic interaction chromatography

Monolithic silica capillary columns for hydrophilic interaction liquid chromatography (HILIC) were prepared by on-column polymerization of acrylic acid on monolithic silica in a fused silica capillary modified with anchor groups. The products maintained the high permeability (K=5 x 10(-14)m(2)) and provided a plate height (H) of less than 10 microm at optimum linear velocity (u) and H below 20 microm at u=6mm/s for polar solutes including nucleosides and carbohydrates. The HILIC mode monolithic silica capillary column was able to produce 10000 theoretical plates (N) with column dead time (t(0)) of 20s at a pressure drop of 20 MPa or lower. The total performance was much higher than conventional particle-packed HILIC columns currently available. The gradient separations of peptides by a capillary LC-electrospray mass spectrometry system resulted in very different retention selectivity between reversed-phase mode separations and the HILIC mode separations with a peak capacity of ca. 100 in a 10 min gradient time in either mode. The high performance observed with the monolithic silica capillary column modified with poly(acrylic acid) suggests that the HILIC mode can be an alternative to the reversed-phase mode for a wide range of compounds, especially for those of high polarity in isocratic as well as gradient elution.     

High temperature vaporizing chambers for large volume GC injections and on-line LC-GC

The suitability of some chambers for sample evaporation at high input flow rates (>100 μl/min) was studied by visual experiments. The chambers were at temperatures far above the solvent boiling point in order to achieve the heat transfer required. Shooting liquid owing to violent evaporation, flooding of the chamber as a result of cooling, and excessively high vapor pressure causing backflow into the gas supply system were found to be the limiting factors. Fused silica capillaries into which a piece of wire or polyimide-free fused silica capillary had been inserted were found to be suitable for the vaporization of “easy” solvents, such as alkanes (up to some 1.7 ml/min), but packed beds were required to achieve favorable evaporation of dichloromethane or methanol/water (up to some 800 and 300 μl/min, respectively).