Injection valve for ultrahigh-pressure liquid chromatography

The increased interest in HPLC at elevated pressures, beyond the conventional 6000 psi (400 bar), has created a demand for injection systems capable of withstanding pressures beyond the 20,000 psi (1380 bar). To achieve high-resolution separations, an appropriate length of columns packed with sub 2-microm packing materials, a 30,000-40,000 psi (2070-2760 bar) pressure range is desirable. A new air-actuated needle valve injection system rated to withstand pressures of up to 40,000 psi (2760 bar) has been evaluated. Under isocratic chromatographic conditions, injecting 200 nL and operated at approximately 20,000 psi (1380 bar), the system showed a peak area reproducibility of approximately 2.5% RSD, contrasting the 5% RSD of a pressured-balanced injection system operated under similar conditions. Programmed for partial loop injections using injection times of 300-700 ms (injection volumes in the range of 1-2.5 microL) and operated at pressures close to 30,000 psi (2070 bar), the reproducibility in peak area for the amounts injected was approximately 1.5% RSD or lower, while an injection time of 100 ms resulted in a reproducibility of 3-4% RSD. The new injection system did not show any significant carryover, and after thousands of injections, the system has not shown sign of wear, loss of pressure during injection, or loss in chromatographic performance.     

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.     

Nanoflow gradient generator for capillary high-performance liquid chromatography

A novel method of generating a nanoflow gradient elution for a capillary high-performance liquid chromatography (HPLC) system has been developed. An important feature of this system is that any gradient (GR) profile generated by a conventional microflow GR pump can be asymptotically traced and converted as a corresponding nanoflow GR profile simply by using a 10-port switching valve with two injection loops installed. Consequently, it has been called an "asymptotic trace 10-port valve" (AT10PV) nanoflow GR generator. Performance of the AT10PV nanoflow GR generator was tested in the range of flow rates from 50 to 500 nL/min. The test demonstrated that the AT10PV nanoflow GR generator can asymptotically trace the original gradient profile with good reproducibility. A capillary HPLC system using the AT10PV nanoflow GR generator provides reasonably good repeatability of peak retention times on the chromatogram of the tryptic digest of a BSA sample, RSD of less than 0.3% at a flow rate of 200 nL/min. It also enables sequential running of a series of sample injections in the same manner as conventional analysis at microflow rates.     

On-line process control of gradient elution liquid chromatography

The typical measure of the stability of analytical HPLC methods in the pharmaceutical laboratory is standards injected repeatedly throughout the sample sequence. To obtain improved control of the analysis and reduction of the number of standards and replicates, a novel approach to treat the analytical run as a process, where the chromatographic data is the product, is proposed. Thus, an alternative and continuous system suitability test procedure is described. This is obtained by continuous monitoring of several parameters of the chromatographic system such as pressure, temperature, and conductivity. The data are analyzed in real time with chemometrics to produce easily interpreted control charts. Gradient elution LC is extensively employed in pharmaceutical analysis. A gradient elution system is inherently dynamic due to the mobile-phase composition being changed during the chromatographic run. To handle the dynamics, suitable chemometric tools are needed. In this report, we extend the use of liquid chromatography process control (LCPC) to gradient elution LC by creating partial least-squares regression batch models of the data collected. The gradient elution LCPC system was evaluated by inducing disturbances, and it was shown to easily detect any real or simulated deviation.     

Determination of lipophilicity by gradient elution high-performance liquid chromatography

A novel method for the determination of lipophilicity using a simple HPLC protocol based on gradient elution chromatography is presented and compared to the common isocratic log k'(w) procedure. Linear relationships with high correlation coefficients between both methods for biologically active nucleosides and cyclic nucleotides as well as for environmentally relevant aromatic hydrocarbons were found. A mathematical fit to support the empirically determined linear relationship is presented. It is shown that the observed relationship between log k'(w) and the apparent capacity factor (k'(g)) determined by gradient elution is derivable by theoretical considerations as well. Since the gradient method is much less time-consuming compared to other procedures, it represents a convenient alternative for determining lipophilicity data in the future.     

Reduction of reequilibration time following gradient elution reversed-phase liquid chromatography

A simple and convenient method for the reduction of column reequilibration time following gradient elution reversed-phase liquid chromatography is described. This method utilizes the addition of a constant volume of 3% 1-propanol to the mobile phase throughout the solvent gradient to provide consistent solvation of the reversed-phase stationary phase. Reductions in reequilibration time of up to 78% have been observed. The effect of alkyl chain bonding density on reequilibration volume is also examined. A maximum in the mobile phase volume necessary to reequilibrate the column is found at a bonding density of about 2.9 mumol/m2. The relationship of reequilibration volume to bonding density supports the partitioning model of retention for reversed-phase liquid chromatography.     

Analytical solutions of the ideal model for gradient liquid chromatography

The analytical solutions of the ideal model for gradient elution that ignores the influence of the solute concentration on the retention factor (k) were studied by using the method of characteristics for solving partial differential equations. It is found for any gradient profiles and solvent strength models used that the concentration of the solute will be discontinuous where the mobile-phase composition is. On a given characteristic curve, the product of the concentration and the retention factor is kept constant at the point where the concentration is continuous. At the point where the concentration is discontinuous, the product on the left side of this point is equal to that on the right side. We also discussed the basic equations to predict the retention time in gradient elution and introduced the injection time into them. For linear solvent strength stepwise and linear gradient elution, general expressions were proposed for the prediction and they can be used as the basis to derive others for specific gradient modes such as single linear, stepwise, and ladderlike gradients. For these modes, simple expressions to account for the band compression and the concentration change during the elution were also given.     

Use of 1.5-microm porous ethyl-bridged hybrid particles as a stationary-phase support for reversed-phase ultrahigh-pressure liquid chromatography

A new ethyl-bridged hybrid packing material was evaluated in terms of its suitability for ultrahigh-pressure liquid chromatography (UHPLC). The 1.5-microm particles were obtained and packed into 30-microm-i.d. fused-silica capillary columns up to 50 cm in length. The particles were evaluated by isocratic reversed-phase UHPLC at pressures up to 4500 bar (65,000 psi). The chromatographic performance of these particles was found to be similar to the performance of 1.0-microm nonporous silica particles. The mechanical strength of the ethyl-bridged hybrid material was evaluated by running a 15-cm-long column at pressures up to 4500 bar. No breakdown of the particles in the packed bed was observed. The sample loading capacity of the hybrid material was evaluated and compared to 1.0-microm nonporous silica material by observing analyte peak width versus amount injected. The observed improvement in loading capacity for the hybrid material versus nonporous silica was consistent with the improvement predicted by comparing the phase ratios of the two materials.     

Linear velocity surge caused by mobile-phase compression as a source of band broadening in isocratic ultrahigh-pressure liquid chromatography

A linear velocity surge caused by mobile-phase compression was investigated as a source of band broadening for ultrahigh-pressure liquid chromatography (UHPLC). To measure the effect of compression on mobile-phase velocity, ionic sample fronts were monitored using a contactless conductivity detector, as they migrated through long packed capillaries. Mobile-phase compression was found to cause an abnormally high linear velocity surge as the pressure was applied to the inlet of the column. An empirical equation was developed to describe the mobile-phase flow velocity during and after compression. Data fits to this equation were then used to determine the amount of additional variance caused by mobile-phase compression. For a 10/90 v/v acetonitrile/water mobile phase, the velocity surge occurred over roughly 10-15% of the column length. In addition, the velocity surge caused by mobile-phase compression was found to be capable of causing a 50% increase in the measured van Deemter C-terms for reversed-phase UHPLC.     

High-speed gas chromatography using synchronized dual-valve injection

A novel injection technique for high-speed gas chromatography is demonstrated. Synchronized dual-valve injection is shown to provide peak widths as low as 1.5 ms (width at half-height) for an unretained analyte. This was achieved using a 0.5-m DB-5 column with an internal diameter of 100 microm and a film thickness of 0.4 microm operated at a temperature of 150 degrees C with a column absolute head pressure of 85 psi, resulting in a dead time of only t(o) = 26 ms ( approximately 1900 cm/s, 26 mL/min). Using the DB-5 column in a 1-m length under the same instrumental parameters, with a resulting linear flow velocity of 935 cm/s (12.7 mL/min, t(o) = 117 ms), a minimum peak width of 3.3 ms was obtained. During an isothermal separation, 10 analytes were separated in a time window of 400 ms. A rigorous comparison of experimental and theoretical band-broadening data based on the Golay equation showed that band broadening is limited almost entirely by the chromatographic band broadening terms expressed by the Golay equation and not by extra column band broadening due to the injection process. Synchronized dual-valve injection offers a rugged and inexpensive design, providing extremely reproducible injections with peak height precision of 2.4% (RSD) and low run-to-run variation in retention times, with an average standard deviation less than 0.1 ms. Herein, synchronized dual-valve injection is demonstrated as a proof of principle using high-speed diaphragm valves. It is foreseen that the injection technique could be readily implemented using a combination of thermal modulation and high-speed valve hardware, thus optimizing the mass transfer and not significantly sacrificing the limit of detection performance for high-speed GC. Further implications are that, if properly implemented, high-speed temperature programming coupled with this new technology should lead to very large peak capacities for approximately 1-s separations.     

Thermal modulation for multidimensional liquid chromatography separations using low-thermal-mass liquid chromatography (LC)

We report on a proof-of-principle experiment with a novel thermal modulation device with potential use in two-dimensional liquid chromatography (LC × LC) systems. It is based on the thermal desorption concept used in two-dimensional gas chromatography (GC × GC) systems. Preconcentration of neutral analytes eluting from the first dimension column is performed in a capillary "trap" column packed with highly retentive porous graphitic carbon particles, placed in an aluminum low-thermal-mass LC heating sleeve. Remobilization of the trapped analytes is achieved by rapidly heating the trap column, by applying temperature ramps up to +1200 °C/min. Compared to the nonmodulated signal, the presented thermal modulator yielded narrow peaks, and a concentration enhancement factor up to 18 was achieved. With a thermally modulated LC separation of an epoxy resin, it is shown that when the thermal modulation is applied periodically, the trapped and concentrated molecules can be released periodically and that the modulating interface can both serve as a preconcentration device and as an injector for the second dimension column of an LC × LC setup. Because of the thermal modulation, a high-molecular-weight epoxy resin could be adequately separated and the different fractions were identified with a GPC analysis, as well as an offline second dimension LC analysis.     

A photovoltaic detection system for flow injection and liquid chromatography

A photoionization system has been developed for flow injection and liquid chromatographic applications that employs photovoltaic rather than traditional photoconductive detection. This detection system utilizes an excimer laser at 248 nm (5.0 eV) to achieve one-photon excitation in alcoholic, aqueous, and ionic solutions. The photovoltaic response is reported for a variety of solutes including aliphatic and aromatic amines, aldehydes, ketones, carboxylic acids, and halogenated compounds. The photovoltaic method is characterized with respect to solute concentration and flow rate. The detection limit is 5 × 10(-)(6) M (2.3 ng) for aniline in methanol with a linear dynamic range greater than 2 orders of magnitude, which compares favorably to UV-visible absorbance detection. Photovoltaic detection is demonstrated for a series of substituted anilines and aldehydes separated by reversed-phase liquid chromatography.     

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.     

Integration of pillar array columns into a gradient elution system for pressure-driven liquid chromatography

A gradient elution system for pressure-driven liquid chromatography (LC) on a chip was developed for carrying out faster and more efficient chemical analyses. Through computational fluid dynamics simulations and an experimental study, we found that the use of a cross-Tesla structure with a 3 mm mixing length was effective for mixing two liquids. A gradient elution system using a cross-Tesla mixer was fabricated on a 20 mm × 20 mm silicon chip with a separation channel of pillar array columns and a sample injection channel. A mixed solution of water and fluorescein in methanol was delivered to the separation channel 7 s after the gradient program had been started. Then, the fluorescence intensity increased gradually with the increasing ratio of fluorescein, which showed that the gradient elution worked well. Under the gradient elution condition, the retention times of two coumarin dyes decreased with the gradient time. When the gradient time was 30 s, the analysis could be completed in 30 s, which was only half the time required compared to that required for an isocratic elution. Fluorescent derivatives of aliphatic amines were successfully separated within 110 s. The results show that the proposed system is promising for the analyses of complex biological samples.     

DNA microsatellite analysis using ion-pair reversed-phase high-performance liquid chromatography

Genotyping based on short tandem repeat (STR) regions is used in human identification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. Analysis of STR systems using slab gel electrophoresis requires lengthy and labor-intensive procedures. Therefore, alternative methods such as capillary electrophoresis or ion-pair reversed-phase high-performance liquid chromatography (IPRP HPLC) have been used to analyze DNA. IPRP HPLC offers an attractive substitute to gel electrophoresis for STR analysis because of the reduced analysis time, and there is no need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for the sizing and typing of STR alleles from the HUMTHO1 locus. The IPRP HPLC conditions (column temperature, flow rate, percent organic modifier per minute) were optimized for the separation of PCR products. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in their native state (double standard) with the use of internal markers. The typing results correlated 100% to accepted methods of DNA typing. The analysis time for the HUMTHO1 locus was less than 14 min, and the alleles could be peak captured for further examination following such as sequencing.     

Universal response in liquid chromatography using charged aerosol detection

A new, empirical approach is introduced to correct for the varying response of aerosol-based detectors with the varying composition of the mobile phase during gradient elution in HPLC. A Corona charged aerosol detector was used in the experiments. The detector is characterized by a nearly universal response at a given, constant mobile-phase composition for sufficiently nonvolatile analytes. A second pump was used to deliver an exactly inverse gradient compared to the analytical HPLC system, and both flows were mixed in a tee piece before introduction to the Corona detector. The approach proposed made it possible to extend the universal response from isocratic to gradient elution conditions in HPLC, vastly improving the usefulness of this detection technique. The constant response of the detector obtained in this way was first demonstrated in flow injection analysis. Very similar calibration curves were obtained for six sulfonamide drugs after mobile-phase compensation. The approach was also applied to gradient elution with excellent results. The data were characterized by good precision ranging from 4% RSD at 10 mg/L to 1.6% RSD at 780 mg/L. The average limit of detection with a 2-microL injection was 0.5 mg/L, corresponding to 1 ng injected on the column. The approach proposed allows quantification of unknown compounds, e.g., in pharmaceutical mixtures. Measurement of analytes at a relative concentration of 0.05% versus the main component is demonstrated.