Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (approximately 1-2 microm) and through-pores (approximately 2-8 microm) in a fused-silica capillary (50-200 microm I.D.). The columns were evaluated in HPLC after derivatization to C18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 microm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (> 80%) and large through-pores resulted in high permeability (K = 8 x 10(-14) -1.3 x 10(-12) m2) that was 2-30 times higher than that of a column packed with 5-mirom silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8-12 microm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-microm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.     

Ultrahigh pressure liquid chromatography using elevated temperature

Fast liquid chromatographic (LC) methods are important for a variety of applications. Reducing the particle diameter (d(p)) is the most effective way to achieve fast separations while preserving high efficiency. Since the pressure drop along a packed column is inversely proportional to the square of the particle size, when columns packed with small particles (<2 microm) are used, ultrahigh pressures (>689 bar) must be applied to overcome the resistance to mobile phase flow. Elevating the column temperature can significantly reduce the mobile phase viscosity, allowing operation at higher flow rate for the same pressure. It also leads to a decrease in retention factor. The advantage of using elevated temperatures in LC is the ability to significantly shorten separation time with minimal loss in column efficiency. Therefore, combining elevated temperature with ultrahigh pressure facilitates fast and efficient separations. In this study, C6-modified 1.0 microm nonporous silica particles were used to demonstrate fast separations using a temperature of 80 degrees C and a pressure of 2413 bar. Selected separations were completed in 30 s with efficiencies as high as 220,000 plates m(-1).     

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.     

Fritless capillary electrochromatography

The preparation of packed capillaries with stable frits of good quality can be a hurdle to obtain efficient separations in capillary electrochromatography (CEC). Especially with particles smaller than 3 microm, frit preparation is cumbersome. Highly efficient separations using packed capillaries without frits are presented. Under appropriate CEC conditions the particles were retained by electrophoretic attraction towards the anode by a tapered capillary inlet, without the need of a frit at the outlet end. Such fritless capillaries, packed with 1.5 microm nonporous reversed-phase particles, allowed separations with efficiencies of more than 500,000 plates/m. Once the capillaries were conditioned properly, more than 100 separations could be performed with good repeatability. With respect to separation efficiency, fritless capillaries packed with 3 microm particles were comparable with standard CEC capillaries with frits. Examples of separations of steroids, a pesticide and its by-products, and cardiac glycosides under various CEC conditions are shown.     

Elevated-temperature ultrahigh-pressure liquid chromatography using very small polybutadiene-coated nonporous zirconia particles

Capillary columns packed with small diameter particles typically lead to low permeability and long separation times in high-performance liquid chromatography. Ultrahigh pressures (>10,000 p.s.i.; 1 p.s.i. is identical with 6,894.76 Pa) can be used to overcome the limitations that small particles impose. Ultrahigh-pressure liquid chromatography (UHPLC) has demonstrated great potential for high-speed and high-efficiency separations. Decreasing the viscosity of the mobile phase by elevating the temperature could additionally reduce the pressure drop and facilitate the use of longer columns or smaller particles to achieve even higher total plate numbers. For this reason, we investigated the use of elevated temperatures in UHPLC. Water-resistant, flexible heater tape covered with insulation was used to provide the desired heat to the column. Polybutadiene-coated 1 microm nonporous zirconia particles were used because of their chemical stability at elevated temperature. A column efficiency as high as 420,000 plates m(-1) was obtained. The effects of temperature and pressure on the separation of parabens were investigated. Separation of five herbicides was completed in 60 s using 26,000 p.s.i. and 90 degrees C.     

Ultrahigh-pressure liquid chromatography using a 1-mm id column packed with 1.5-microm porous particles

The evolution of chromatography has led to the reduction in the size of the packing materials used to fabricate HPLC columns. The increase in the backpressure required has led to this technique being referred to as ultrahigh-pressure liquid chromatography (UHPLC) when the column backpressure exceeds 10000 psi (approximately 700 bar). Until recently, columns packed with sub-2-microm materials have generally fitted into two classes; either short (less than 5 cm) columns designed for use on traditional HPLC systems at pressures less than 5000 psi (350 bar), or capillary columns (inner diameters less than 100 microm). By using packing materials with diameters <2 microm to fabricate UHPLC columns, there is an increase in efficiency and a decrease in the analysis time that are directly proportional to the size of the packing material. In order to realize and exploit the increase in efficiency, however, the columns must maintain lengths typically associated with analytical columns (15-25 cm). We have packed 1 mm diameter, 150 mm in length columns with 1.5 microm packing material, and evaluated their performance in UHPLC. The pressure required to achieve optimum linear velocities in plots of plate height versus linear velocity was in the vicinity of 1104 bar (16000 psi). The 1.5 microm particle-packed column was compared with the more traditional 150 mm long analytical columns packed with 3 microm materials. This column showed an efficiency that was approximately twice that observed with the 3 microm packed column and a concomitant reduction in the analysis time, theoretically predicted.     

Mesoporous polybutadiene-modified zirconia for high-temperature packed capillary liquid chromatography: column preparation and temperature programming stability

In the present study, three different methods for packing of 3 microm PBD-ZrO2 particles in 0.5 mm i.d. glass-lined stainless steel columns have been examined. The two first methods were based on a traditional downstream high-pressure technique using tetrachloromethane (Method I) or aqueous Triton X-100 (Method II) as slurry solvents, while Method III was an upstream high-pressure flocculating method with stirring, using isopropanol both as the slurry and packing solvent. Method I was found to be superior in terms of efficiency, producing 0.5 mm i.d. x 10 cm columns with almost 90,000 plates m(-1) for toluene (R.S.D. = 8.7%, n = 3), using a slurry concentration of 600 mg ml(-1), ACN-water (50:50 (v/v)) as the packing solvent and a packing pressure of 650 bars. For Method I, the slurry concentration, column i.d., column length and initial packing pressure were found to have a significant effect on column efficiency. Finally, the long-term temperature stability of the prepared columns was investigated. In isothermal mode, using ACN-20 mM phosphate buffer, pH 7 (50:50 (v/v)) as the mobile phase, the columns were found to be stable for at least 3,000 void volumes at 100 degrees C. At this temperature, the solute efficiencies changed about 5-18% and the retention factors changed about 6-8%. In temperature programming mode (not exceeding 100 degrees C), on the other hand, a rapid decrease in both column efficiency and retention factors was observed. However, when the columns were packed as initially described, ramped up and down from 50 to 100 degrees C for 48 h and refilled, fairly stable columns with acceptable efficiencies were obtained. Although not fully regaining their initial efficiency after refilling, the solute efficiencies changed about 19-28% (32-37%) and the retention factors changed about 4-5% (13-17%) after running 3,000 (25,000) void volumes or 500 (3,900) temperature programs.     

Characterisation of RPLC columns packed with porous sub-2 microm particles

Eight commercially available sub-2 microm octadecyl silane columns (C18 columns) have been characterised by the Tanaka protocol. The columns can be grouped into two groups that display large differences in selectivity and peak shape due to differences in hydrophobicity, degree of surface coverage and silanol activity. Measurements of particle size distributions were made using automated microscopy and electrical sensing zone measurements. Only a weak correlation could be found between efficiency and particle size. Large differences in column backpressure were observed. These differences are not related to particle size distribution. A more likely explanation is differences in packing density. In order to take full advantage of 100-150 mm columns packed with sub-2 microm particles, it is often necessary to employ not only an elevated pressure but also an elevated temperature. A comparison between columns packed with sub-2, 3 and 5 microm versions of the same packing indicates potential method transferability problems for several of the columns due to selectivity differences. Currently, the best alternative for fast high-resolution LC is the use of sub-2 microm particles in combination with elevated pressure and temperature. However, as shown in this study additional efforts are needed to improve transferability as well as column performance.     

In-line coupling of low-pressure short capillary electrochromatography columns to electrospray ionisation mass spectrometry

A new in-house designed and constructed injection valve for capillary electrochromatography (CEC) based on a rotating injection part with compartments for the eluent as well as for the sample has been coupled to a mass spectrometer via a sheath flow electrospray ionisation (ESI) interface, using short capillary columns of 15 cm length. The CEC columns were packed with 3 microm C(18) bonded silica particles, and a mixture of peptides was analysed using an ammonium acetate/acetonitrile eluent. A significant increase in the signal-to-noise ratio was obtained when the peptides were dissolved in water with the same content of organic modifier as in the eluent with an addition of 0.5% (v/v) acetic acid. When the CEC analysis was performed without any additional pressure, the separation current sometimes dropped tremendously due to bubble formation, caused by different permeability in the first and packed part of the column causing an extremely low electroosmotic flow. The separation current was restored to its original value by applying only 7 bar at the inlet of the CEC column, and the separation performance for the test peptides was recovered. A comparison of the CEC performance of peptides in pure CEC mode and in low-pressure CEC mode is reported.     

Development of a supercritical fluid chromatography high-resolution separation method suitable for pharmaceuticals using cyanopropyl silica

A method has been developed for the analysis of a broad spectrum of pharmaceuticals using packed column supercritical fluid chromatography (pSFC) on a cyanopropyl silicagel stationary phase. Five 25 cm x 4.6 mm I.D., 5.0 microm columns were coupled to generate ca. 100000 plates. The selectivity was tuned by varying the nature and concentration of various modifiers and additives in the carbon dioxide mobile phase. It was noted that pressure influences both efficiency and selectivity of the chromatographic process. Final method conditions are: outlet pressure 100 bar, flow 2.0 mL/min, temperature 40 degrees C, organic modifier program from 5% (1 min) to 40% at 2.0%/min, organic modifier composition methanol:acetonitrile in a ratio of 3:1 (variable according to sample composition) with peak symmetry additives trifluoroacetic acid and diisopropylamine both at levels of 0.5%.     

Comparison of the efficiency of microparticulate and monolithic capillary columns

A comparison is made between the efficiency of microparticulate capillary columns and silica and polymer-based monolithic capillary columns in the pressure-driven (high-performance liquid chromatography) and electro-driven (capillary electrochromatography) modes. With packed capillary columns similar plate heights are possible as with conventional packed columns. However, a large variation is observed in the plate heights for individual columns. This can only be explained by differences in the quality of the packed bed. The minimum plate height obtained with silica monolithic capillary columns in the HPLC mode is approximately 10 microm, which is comparable to that of columns packed with 5-microm particles. The permeability of wide-pore silica monoliths was found to be much higher than that of comparable microparticulate columns, which leads to much lower pressure drops for the same eluent at the same linear mobile phase velocity. For polymer-based monolithic columns (acrylamide, styrene/divinyl benzene, methacrylate, acrylate) high efficiencies have been found in the CEC mode with minimum plate heights between 2 and 10 microm. However, in the HPLC mode minimum plate heights in the range of 10 to 25 microm have been reported.     

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.     

Capillary liquid chromatography and capillary electrochromatography using silica hydride stationary phases

A hydride-based octadecyl stationary phase on both 4.0 and 1.8 microm silica particles is tested in both the capillary LC and the pressurized capillary electrochromatography (pCEC) modes. These two materials are compared to standard C18 stationary phase made by organosilanization and to the hydride material packed into a convention 4.6mm I.D. column. The performance of the capillary columns is evaluated in terms of analysis times for various mixtures as well as efficiency. Of particular interest are the differences between the LC mode where only laminar flow is present and pCEC operation where a flat electrodriven flow profile is superimposed on the parabolic pressurized flow. Differences in performance between columns packed with 4.0 and 1.8 microm particle silica are also evaluated.     

Possibilities and limitations of fast GC with narrow-bore columns

In this work, two narrow-bore capillary columns with different internal diameters (I.D.) 0.15 mm (15 m length, 0.15 microm film thickness) and 0.10 mm (10 m length, 0.10 microm film thickness) with the same stationary phase (5% diphenyl 95% dimethylsiloxane), phase ratio and separation power were compared with regard to their advantages, practical limitations and applicability in fast GC on commercially available instrumentation. The column comparison concerns fast GC method development, speed and separation efficiency, the sample transfer into the column utilizing split and splitless inlet, sample capacity, detection (analysing compounds of a wide range of polarities and volatilities--even n-alkanes C16-C28 and selected pesticides) and ruggedness (in the field of ultratrace analysis of pesticide residues in real matrix). Under conditions corresponding to speed/separation efficiency trade-off 0.10 mm I.D. versus 0.15 mm I.D. column provides a speed gain of 1.74, but all other parameters investigated were better for the 0.15 mm I.D. column concerning more efficient sample transfer from inlet to the column using splitless injection, no discrimination with split injection. Better sample capacity (three times higher for the 0.15 mm than for the 0.10 mm I.D. column) resulted in improved ruggedness and simpler fast GC-MS method development.     

Very high-pressure capillary liquid chromatography assisted by voltage

Capillary liquid chromatography at moderately high pressures and capillary electrochromatography (CEC) have been combined to drive the mobile phase through capillary columns packed with small diameter particles. In a column packed with 1.5 microm nonporous particles, linear velocities near 3mm/s were observed when combining inlet pressures of 690 bar (10,000 psi) and an applied voltage of 25 kV. Optimum linear velocity for the column was achieved using a pressure-voltage combination of 350 bar (5000 psi) and 5 kV. Separation efficiencies at near optimum linear velocity agreed with those predicted by the van Deemter equation for liquid chromatography. Retention factors were observed to decrease under pressure-voltage combination as the voltage was increased; such a behavior has been attributed to Joule heating effects.     

Semi-micro size-exclusion chromatography of polymers : Calibration of columns and comparison with conventional columns

Stainless-steel tubes having inside diameters of 1.5 mm and 1.8 mm were packed with polystyrene gels of particle diameter 10 ± 2 μm. Two 50 cm × 1.8 mm I.D. packed columns, connected in series, were calibrated and molecular-weight averages of polystyrene NBS 706 were measured, the results coinciding with the data of the National Bureau of Standards. The peak widths of polystyrenes of narrow molecular-weight distributions in both semi-micro column (four 25 cm × 1.5 mm I.D.) and conventional column (two 50 cm × 8 mm I.D.; packed by the manufacturer) systems were determined at different mobile-phase velocities, and the minimum peak width in the latter system was obtained at the velocity of 0.2 mm/sec, which was higher than that for the semi-micro system. The interstitial volume was higher and the inner volume was lower for the semi-micro column system (1.8 mm I.D.) than those for the conventional one, which means that semi-micro columns were packed less densely, resulting in a steep calibration curve. The peak height of a solute was proportional to the cell length of an ultraviolet detector if the sample load was proportional to the cross-sectional areas of columns having the same column efficiency. Although conventional size-exclusion chromatography has many advantages in respect of velocity, calibration curtve and sample peak height, semi-micro size-exclusion chromatography still holds some merits such as low consumption of gels and of mobile-phase solvents.     

Axial development and radial non-uniformity of flow in packed columns

Flow inhomogeneity and axial development in low-pressure chromatographic columns have been studied by magnetic resonance imaging velocimetry. The columns studied included (a) an 11.7-mm I.D. column packed with either 50 microm diameter porous polyacrylamide, or 99 or 780 microm diameter impermeable polystyrene beads, and (b) a 5-mm I.D. column commercially packed with 10 microm polymeric beads. The packing methods included gravity settling, slurry packing, ultrasonication, and dry packing with vibration. The magnetic resonance method used averaged apparent fluid velocity over both column cross-sections and fluid displacements greater than one particle diameter and hence permits assessment of macroscopic flow non-uniformities. The results confirm that now non-uniformities induced by the conical distributor of the 11.7-mm I.D. column or the presence of voids at the column entrance relax on a length scale of the column radius. All of the 11.7-mm I.D. columns examined exhibit near wall channeling within a few particle diameters of the wall. The origins of this behavior are demonstrated by imaging of the radial dependence of the local porosity for a column packed with 780 microm beads. Columns packed with the 99-microm beads exhibit reduced flow in a region extending from ten to three-to-five particle diameters from the wall. This velocity reduction is consistent with a reduced porosity of 0.35 in this region as compared to approximately 0.43 in the bulk of the column. Ultrasonicated and dry-packed columns exhibit enhanced flow in a region located between approximately eight and 20 particle diameters from the wall. This enhancement maybe caused by packing density inhomogeneity and/or particle size segregation caused by vibration during the packing process. No significant non-uniformities on length scales of 20 microm or greater were observed in the commercially packed column packed with 10 microm particles.     

On-line preconcentration of trace carcinogenic polycyclic aromatic hydrocarbons (PAHs) in microcolumn liquid chromatography via large volume injection

The ability and efficiency of micro precolumns made of C30 particles, monolithic silica C18 stationary phase and quartz wool coated with C30, which act as novel solid phase absorbing materials, for the on-line enrichment of aqueous polycyclic aromatic hydrocarbons (PAHs) in microcolumn liquid chromatography (LC) was investigated. The enrichment unit was designed in such a way that micro precolumns were directly connected to a 6-port micro injection valve via fused-silica tubing (0.05 mm I.D.) in order to minimize band broadening of the samples, and the enrichment efficiency of the three materials was tested using 14 PAHs, which are selected by the US Environmental Protection Agency (US EPA), as the analytes. The separation of PAHs was evaluated by using laboratory-made C30 or ODS capillary columns and the results were compared. There were no significant differences showed from the separation of PAHs in terms of peak signal between the C30 and ODS capillary columns, but the C30 capillary column was chosen for the following experiment due to its ability to produce better repeatability than the ODS column. By using the three kinds of precolumn materials, results showed that the precolumn packed with C30 particles as well as the capillary monolithic C18 precolumns (0.1 or 0.2 mm I.D.) provided better recovery than those of the quartz wool's. As long as the recovery and separation of the PAHs were concerned, 0.1 mm I.D. monolithic C18 precolumn showed the best results and the R.S.D.s (N = 7) for the retention time, peak area and peak height were between 0.70-1.5, 2.3-5.8 and 2.4-6.6%, respectively. Large volume injection up to 0.5 mL, i.e. 2500-fold enrichment, was possible and no negative effect on the separation profile was found. The LOD (S/N = 3) were between 0.10 and 4.6 pg mL⁻¹, while the LOQ (S/N = 10) were in the range of 0.32-15 pg mL⁻¹, which showed that the system is comparable to many major analytical techniques and is sensitive enough for the trace analysis of PAHs in environmental samples. The system was then applied to the determination of trace PAHs present in soil sample which was randomly taken from a nearby highway.     

Electrochromatography with a 2.7 mm inner diameter monolithic column

Monolithic columns of 2.7 mm I.D. have been prepared and used in electrochromatography (EC) separation. Although capillary electrochromatography (CEC) has higher separation efficiency, it displays some shortcomings, such as limited sample loadability and restricted concentration detectability etc. In this paper, we investigate the feasibility of EC separation with millimeter diameter monolithic columns. By using a designed preparation method of monolithic column packed with about 150 microm quartz sand, the effect of Joule heating can be reduced, and the processes of frit making and column packing can be avoided. The concentration detectability of the EC is improved comparing with that of CEC. Moreover, the separation efficiency of 52,000 plates/m was achieved with a 70 mm length and 2.7 mm I.D. monolithic column.     

Fast and high-resolution ion chromatography at high pH on short columns packed with 1.8 microm surfactant coated silica reverse-phase particles

Rapid ion chromatographic separations of small inorganic anions are performed on columns packed with high-pH resistant Zorbax Extend-C18 1.8 microm silica particles. Seven anions (iodate, chloride, nitrite, bromide, nitrate, phosphate, sulphate) are separated with 1.3 and 2 cm long x 0.46 cm I.D. C18 columns coated with the surfactant didodecyldimethylammonium bromide (DDAB). A 40 s separation is achieved at 2 mL/min with a 2.5 mM 4-hydroxybenzoic acid eluent at pH 10. Finally, the DDAB removal procedure is improved to eliminate the pressure build-up caused by precipitation of the surfactant in the column upon uncoating.