A novel switchable water stationary phase for supercritical fluid chromatography.

A novel pH switchable water stationary phase is presented for use in supercritical fluid chromatography (SFC). By adding NH4OH to the water coating and system hydration, changes in CO2 pressure and temperature allow a wide range of stationary phase pH conditions (∼3-9) to be achieved, which impact analyte retention properties. For example, 100 atm and 50 °C produces an acidic water stationary phase (pH near 4.0) where octanoic acid readily elutes while the base caffeine does not. Conversely, at 80 atm and 120 °C a basic water stationary phase (pH near 8.0) is obtained and the opposite occurs. Further, under constant pressure and temperature conditions, simply adding or removing NH4OH from the system is also found to readily allow switching between the basic and acidic water stationary phase modes and demonstrates control over ionizable analyte elution. For instance, hexanoic acid elution is near 40 times more delayed on a basic water stationary phase and, as such, it can be eluted at later points in time as desired by removing the NH4OH and switching to an acidic stationary phase. Experiments indicate that stationary phase pH switching occurs uniformly across the 15 m column length within about 18 s and that analyte retention times are very reproducible upon performing a switch (1.4% RSD; n = 3). Results demonstrate the selectivity factor between acidic and neutral analytes can be reversed and increased about 35 times, while in other trials resolution also similarly increased near 40-fold. By rapidly switching the stationary phase pH back and forth between acidic and basic modes, the selectivity between ionizable analytes could also be increased as desired. Various applications with the system show that it can vastly increase the separation between target analytes and matrix components as required by the dynamics of a particular separation.

Sulfolane as a novel stationary phase for analytical separations by gas chromatography.

Sulfolane is explored as a novel stationary phase for use in analytical separations by capillary column gas chromatography with flame ionization detection (GC-FID). Stainless steel capillaries were found to provide a good substrate for coating and retaining a sulfolane phase, whereas fused silica tubing did not perform well for this. In general, the phase was found to be stable for several hours of use when using elevated carrier gas pressures (90 psi) and a small restriction (25 µm I.D. tubing) at the outlet. This normally provided good performance at temperatures up to about 200 °C with very little background interference in the FID. Given its separation properties, a short 2 m × 100 µm I.D. column was found to be preferable for most separations in this study. Measurements indicated the coating procedure yielded a sulfolane film near 4 µm thick on this column, which produced 4400 plates for benzene with a sample capacity near 30 µg. The sulfolane phase yielded good retention and peak shape for many analytes including alkanes, aromatics, alcohols, bases, sulfides, phosphites, thiols, and others. Compared to longer conventional GC columns, the relatively short sulfolane column was found to offer improved selectivity in the separation of unsaturated, aromatic, and alkane test analytes. As such the method was successfully applied to the analysis of aromatics in gasoline headspace. Results suggest that sulfolane could be a potentially useful stationary phase to further explore in GC separations.

Active control of selectivity in organic acid analysis by gas chromatography.

A new method that allows organic acid selectivity to be dynamically controlled during gas chromatography (GC) is presented. It employs dual in-series stainless steel columns, each coated with a pH-adjusted water stationary phase. The first is a 2 m column coated with a pH 11.4 phase that is connected to a second 11 m column coated with a pH 2.2 phase. In this arrangement, organic acids within sample mixtures are trapped on the first column, while the remaining non-ionizable components continue to separate and elute in the system. Later, by injecting a volatile formic acid solution, the trapped acids are released in-situ to the second column for separation and analysis as desired. The method provides good reproducibility with analyte retention times in consecutive trials yielding an average RSD of 1.9%. Further, depending on column temperature, analytes can be readily retained for periods investigated up to about 30 min without significant deterioration in peak shape. This feature provides considerable control over analyte selectivity and resolution compared to conventional separations. Further, by adding a third conventional GC column in-series, both typical hydrocarbon and enhanced organic acid separations are made possible. The method is applied to the analysis of complex mixtures and matrix interference is found to be significantly minimized. Results indicate that this approach offers beneficial advantages for the selective GC analysis of such acidic analytes.

Dynamic Control of Gas Chromatographic Selectivity during the Analysis of Organic Bases.

A novel method for controlling selectivity during the gas chromatographic (GC) analysis of organic bases is presented. The technique employs tandem stainless steel capillary columns, each coated with a pH adjusted water stationary phase. The first is a 0.5 m trap column coated with a pH 2.2 phase, while the second is an 11 m analytical column coated with a pH 11.4 phase. The first column traps basic analytes from injected samples, while the remaining components continue to elute and separate. Then, upon injection of a volatile aqueous ammonia solution, the basic analytes are released as desired to the analytical column where they are separated and analyzed. Separations are quite reproducible and demonstrate an average RSD of 1.2% for analyte retention times in consecutive trials. Using this approach, the retention of such analytes can be readily controlled and they can be held in the system for periods of up to 1 h without significant erosion of peak shape. As such, it can provide considerable control over analyte selectivity and resolution compared to conventional separations. Further, by employing a third conventional GC column to the series, both traditional hydrocarbon and enhanced organic base separations can be performed. The method is applied to the analysis of complex mixtures, such as gasoline, and much less matrix interference is observed as a result. The findings indicate that this approach could be a useful alternative for analyzing such samples.

Retention Characteristics of a pH Tunable Water Stationary Phase in Supercritical Fluid Chromatography.

The retention characteristics of a novel pH tunable water stationary phase are presented. The method utilizes a change in mobile phase from N2 to CO2 to acidify the water phase in situ and control the ionization and elution of organic acids. With N2 present the phase pH > 5.4 and the acids are ionized and strongly retained. Conversely, with CO2 present the pH < 3.8 and the acids are neutralized and can elute. This effect is reasonably independent of time. For example, at 80°C hexanoic acid readily elutes from a 10 m column after switching to CO2 at any point over a 1 h period. Beyond this, however, some broadening and peak erosion is noted. Acids are also retained on 10 and 2 m columns similarly, since their elution primarily depends upon the change in stationary phase pH. Altering the CO2 solubility in the water phase alone (i.e., through changing system temperature and pressure without using N2) also produces similar changes in stationary phase acidity. However, this approach yields greater system noise and instability. The N2/CO2 switching mode is used to analyze organic acids in various samples and is found to provide high selectivity for them over other matrix components. Therefore, this approach can potentially simplify the analysis of such acids in complex samples.

Capillary gas chromatographic separation of organic bases using a pH-adjusted basic water stationary phase.

The use of a pH-adjusted water stationary phase for analyzing organic bases in capillary gas chromatography (GC) is demonstrated. Through modifying the phase to typical values near pH 11.5, it is found that various organic bases are readily eluted and separated. Conversely, at the normal pH 7 operating level, they are not. Sodium hydroxide is found to be a much more stable base than ammonium hydroxide for altering the pH due to the higher volatility and evaporation of the latter. In the basic condition, such analytes are not ionized and are observed to produce good peak shapes even for injected masses down to about 20ng. By comparison, analyses on a conventional non-polar capillary GC column yield more peak tailing and only analyte masses of 1µg or higher are normally observed. Through carefully altering the pH, it is also found that the selectivity between analytes can be potentially further enhanced if their respective pKa values differ sufficiently. The analysis of different pharmaceutical and petroleum samples containing organic bases is demonstrated. Results indicate that this approach can potentially offer unique and beneficial selectivity in such analyses.

Packed column supercritical fluid chromatography using stainless steel particles and water as a stationary phase.

Stainless steel (SS) particles were demonstrated as a novel useful support for a water stationary phase in packed column supercritical fluid chromatography using a CO2 mobile phase. Separations employed flame ionization detection, and the system was operated over a range of temperatures and pressures. Retention times reproduced well with RSD values of 2.6% or less. Compared to analogous separations employing a water stationary phase coated onto a SS capillary column, the packed column method provided separations that were about 10× faster, with nearly 8-fold larger analyte retention factors, while maintaining good peak shape and comparable column efficiency. Under normal operating conditions, the packed column contains about 131 ± 4 µL/m of water phase (around a 5% m/m coating), which is over 25× greater than the capillary column and also affords it a 20-fold larger sample capacity. Several applications of the packed column system are examined, and the results indicate that it is a useful alternative to the capillary column mode, particularly where analyte loads or sample matrix interference is a concern. Given its high sample capacity, this packed column method may also be useful to explore on a more preparative scale in the future.

An improved multiple flame photometric detector for gas chromatography.

An improved multiple flame photometric detector (mFPD) is introduced, based upon interconnecting fluidic channels within a planar stainless steel (SS) plate. Relative to the previous quartz tube mFPD prototype, the SS mFPD provides a 50% reduction in background emission levels, an orthogonal analytical flame, and easier more sensitive operation. As a result, sulfur response in the SS mFPD spans 4 orders of magnitude, yields a minimum detectable limit near 9×10(-12)gS/s, and has a selectivity approaching 10(4) over carbon. The device also exhibits exceptionally large resistance to hydrocarbon response quenching. Additionally, the SS mFPD uniquely allows analyte emission monitoring in the multiple worker flames for the first time. The findings suggest that this mode can potentially further improve upon the analytical flame response of sulfur (both linear HSO, and quadratic S2) and also phosphorus. Of note, the latter is nearly 20-fold stronger in S/N in the collective worker flames response and provides 6 orders of linearity with a detection limit of about 2.0×10(-13)gP/s. Overall, the results indicate that this new SS design notably improves the analytical performance of the mFPD and can provide a versatile and beneficial monitoring tool for gas chromatography.

Coating properties of a novel water stationary phase in capillary supercritical fluid chromatography.

The coating properties of a novel water stationary phase used in capillary supercritical fluid chromatography were investigated. The findings confirm that increasing the length or internal diameter of the type 316 stainless-steel column used provides a linear increase in the volume of stationary phase present. Under normal operating conditions, results indicate that about 4.9 ± 0.5 µL/m of water phase is deposited uniformly inside of a typical 250 µm internal diameter 316 stainless-steel column, which translates to an area coverage of about 6.3 ± 0.5 nL/mm(2) regardless of dimension. Efforts to increase the stationary phase volume present showed that etching the stainless-steel capillary wall using hydrofluoric acid was very effective for this. For instance, after five etching cycles, this volume doubled inside of both the type 304 and the type 316 stainless-steel columns examined. This in turn doubled analyte retention, while maintaining good peak shape and column efficiency. Overall, 316 stainless-steel columns were more resistant to etching than 304 stainless-steel columns. Results indicate that this approach could be useful to employ as a means of controlling the volume of water stationary phase that can be established inside of the stainless-steel columns used with this supercritical fluid chromatography technique.

Properties of water as a novel stationary phase in capillary gas chromatography.

A novel method of separation that uses water as a stationary phase in capillary gas chromatography (GC) is presented. Through applying a water phase to the interior walls of a stainless steel capillary, good separations were obtained for a large variety of analytes in this format. It was found that carrier gas humidification and backpressure were key factors in promoting stable operation over time at various temperatures. For example, with these measures in place, the retention time of an acetone test analyte was found to reduce by only 44s after 100min of operation at a column temperature of 100°C. In terms of efficiency, under optimum conditions the method produced about 20,000 plates for an acetone test analyte on a 250µm i.d.×30m column. Overall, retention on the stationary phase generally increased with analyte water solubility and polarity, but was relatively little correlated with analyte volatility. Conversely, non-polar analytes were essentially unretained in the system. These features were applied to the direct analysis of different polar analytes in both aqueous and organic samples. Results suggest that this approach could provide an interesting alternative tool in capillary GC separations.

Properties of a novel linear sulfur response mode in a multiple flame photometric detector.

A new linear sulfur response mode was established in the multiple flame photometric detector (mFPD) by monitoring HSO* emission in the red spectral region above 600nm. Optimal conditions for this mode were found by using a 750nm interference filter and oxygen flows to the worker flames of this device that were about 10mL/min larger than those used for monitoring quadratic S2* emission. By employing these parameters, this mode provided a linear response over about 4 orders of magnitude, with a detection limit near 5.8×10(-11)gS/s and a selectivity of sulfur over carbon of about 3.5×10(3). Specifically, the minimum detectable masses for 10 different sulfur analytes investigated ranged from 0.4 to 3.6ng for peak half-widths spanning 4-6s. The response toward ten different sulfur compounds was examined and produced an average reproducibility of 1.7% RSD (n=10) and an average equimolarity value of 1.0±0.1. In contrast to this, a conventional single flame S2* mode comparatively yielded respective values of 6.7% RSD (n=10) and 1.1±0.4. HSO* emission in the mFPD was also found to be relatively much less affected by response quenching due to hydrocarbons compared to a conventional single flame S2* emission mode. Results indicate that this new alternative linear mFPD response mode could be beneficial for sulfur monitoring applications.

Characterization of low-temperature cofired ceramic tiles as platforms for gas chromatographic separations.

A gas chromatography (GC) column is fabricated within a low-temperature cofired ceramic (LTCC) tile, and its analytical properties are characterized. By using a dual-spiral design, a 100 µm wide square channel up to 15 m in length is produced within an 11 cm × 5.5 cm LTCC tile. The channel is dynamically coated with an OV-101 stationary phase that is cross-linked with dicumyl peroxide. While the uncoated LTCC tiles were able to separate a mixture of n-alkanes, the peak shapes were broad (base width of ~2 min) and tailing. In contrast to this, the coated LTCC tiles produced sharp (base width of ~8-10 s), symmetrical, well-resolved peaks for the same analytes. By using a 7.5 m long channel, about 15,000 plates were obtained for a dodecane test analyte. Further, the coated LTCC tiles were found to produce plate heights that were about 3-fold smaller than those obtained from a conventional capillary GC column of similar length, dimension, and coating operated under the same conditions. As a result, test analyte separations were slightly improved in the LTCC tiles, and their overall performance fared well. In terms of temperature programming, it was found that a series of n-alkanes separated on the LTCC tile provided a cumulative peak capacity of around 54 peaks when using C8 to C13 as analyte markers. Results indicate that LTCC tiles provide a viable and useful alternative platform for performing good quality GC separations.

Characterization of the Subcritical Water Extraction of Fluoxetine-Hydrochloride.

The characteristics of using Subcritical Water Extraction (SWE) to recover Fluoxetine-Hydrochloride from both standard solutions and the contents of commercial capsule formulations were investigated. Analysis of solutions and extracts was done by HPLC with UV detection at 254 nm. Standard solutions of Fluoxetine-Hydrochloride were exposed to a variety of SWE operating conditions, including temperatures from 125 to 275°C and periods ranging from 5 to 30 min. Fluoxetine-Hydrochloride could be quantitatively recovered from standard solutions (1.0mg/mL) that were heated up to 175°C for 30 min, up to 200°C for 15 min, or up to 225°C for 10 min. At higher temperatures and/or times, Fluoxetine-Hydrochloride recoveries were generally incomplete and often produced decomposition by-products during the process. By comparison, the concentration of Fluoxetine-Hydrochloride in the standard solution had relatively little effect on recovery. Considering these parameters, an SWE method was developed to extract Fluoxetine-Hydrochloride from the contents of Prozac(®) capsules. It was found that Fluoxetine-Hydrochloride could be quantitatively extracted from the capsule contents in 8 min at a temperature of 200°C using 3.5 mL of water as the extraction solvent. Gelatinization of the starch excipient in the capsule contents was also observed to occur temporarily during the capsule extractions, before ultimately disappearing again. The period of this phenomenon was dependent on both temperature and sample size. The results indicate that SWE can be a very useful method for Fluoxetine-Hydrochloride extraction and suggest that it may be interesting to explore other pharmaceuticals using this method as well.

Increased flow rate compatibility for universal acoustic flame detection in liquid chromatography.

The liquid chromatography (LC) flow rate tolerance of the universal Acoustic Flame Detector (AFD) is characterized and significantly expanded through using larger bore burners. For example, increasing the burner i.d. from 1.00 to 4.00 mm increases the AFD upper flow rate limit from 20 to 100 µL/min. While signal and noise each reduce as the burner i.d. widens, the best current performance is obtained with a 2.30 mm i.d. burner. This approach also allows AFD operation over a broader range of mobile phase temperatures. As a result, the overall increased flow rate compatibility of the detector can facilitate improved chromatography and further development of LC-AFD applications.

Chromatography using a water stationary phase and a carbon dioxide mobile phase.

A novel chromatographic separation method is introduced which employs water (saturated with CO(2)) as a stationary phase and CO(2) (saturated with water) as a mobile phase. Since water and CO(2) have little miscibility, conditions can be attained that create a stationary phase of water lining the inside of an uncoated stainless steel capillary. Because altering temperature and pressure can change both the density of the mobile phase and the polarity of the stationary phase, these experimental parameters offer good flexibility for optimizing separations and allow for different gradient programmed separation options. Further, since this method is free of organic stationary and mobile phase components, it is environmentally compatible and allows the use of universal flame ionization detection. This system offers very good sample capacity, peak symmetry, and retention time reproducibility (∼1% RSD run-to-run, ∼4% RSD day-to-day). Analytes such as alcohols, carboxylic acids, phenols, and tocopherols are employed to investigate this relatively inexpensive and robust method. As an application, the system is used to quantify ethanol in alcoholic beverages and biofuel and to analyze caffeine levels in drinks. In all cases, quantitative results are obtained with quick throughput times and often little need for sample preparation.

Quenching-resistant multiple micro-flame photometric detector for gas chromatography.

A multiple flame photometric detector (mFPD) based on many flames operated in series is introduced for the detection of sulfur and phosphorus compounds. The method employs attributes of a previously developed micro counter-current flame technique to readily establish any number of very small compact flames inside a narrow quartz tube. Results show for the first time that a five flame mFPD mode can improve hydrocarbon quenching resistance nearly 20-fold relative to a single flame (i.e., conventional FPD) mode, and nearly 10-fold relative to a two flame (i.e., dual FPD) mode. Under these conditions, the five flame mFPD mode is shown to maintain about 60% of its original analyte chemiluminescence even in the presence of over 100 mL/min of methane flow into the detector. In contrast to a conventional dual FPD device, the five flame mFPD mode also provides analyte sensitivity that is similar to a conventional FPD. Of note, the mFPD yields minimum detectable limits for sulfur and phosphorus of 4 x 10(-11) g S/s and 3 x 10(-12) g P/s respectively. Analyte selectivity over hydrocarbons, signal reproducibility, and response equimolarity are also improved in the mFPD, making it a potentially useful detector for applications in gas chromatography.

Dynamic control of split flow in packed column supercritical fluid chromatography using dual resistively heated restrictors.

Remote control of the vent/detector split flow ratio in packed column supercritical fluid chromatography (pSFC) with flame ionization detector (FID) is demonstrated using a dual heated restrictor method. Restrictors stemming from a Tee at the separation column outlet were, respectively, fixed into an FID and a vent port, and their individual temperatures were controlled using resistively heated wires. Subsequently, both system pressure and split flow could be manipulated. For example, for applied restrictor temperatures examined up to 600 degrees C, corresponding vent/FID split flow ratios between 2 and 7 were observed depending on the port heated. As well, column pressures around 16-23 MPa were also achievable over the same range. Conversely, isobaric altering of the split flow ratio was possible when opposing positive and negative temperature gradients were applied at the two restrictors. Under these conditions, the system pressure varied less than 1% RSD over a 10 min period. As an application, the method was used to establish stable detector operation in the analysis of n-alkanes under pSFC-FID conditions that initiated flame instability. Results indicate that this technique could be a relatively simple and inexpensive means of controlling system pressure and detector split flow ratios in pSFC-FID.

Carbon dioxide modified subcritical water chromatography.

A novel method of increasing the elution strength in subcritical water chromatography (SWC) by adding CO2 to the water mobile phase is presented. Since the polarity of water reduces dramatically with increasing temperature, this property is used in SWC to create an isocratic mobile phase with tunable elutropic strength in reversed-phase separations. Unfortunately, thermal stability of the stationary phase dictates the upper temperature limit and therefore also the minimum available mobile phase polarity. As a result SWC is often not very effective at eluting non-polar analytes. However, when CO2 is blended into subcritical water, a considerable reduction in mobile phase polarity results and improves such separations. For example, in conventional SWC 1-octanol is not observed to elute from a PRP-1 column after several hours at the maximum column temperature of 200 degrees C. In contrast to this, when CO2 is present at 180atm (1atm=101325Pa) in the mobile phase, 1-octanol elutes with good peak shape in less than 4min at only 100 degrees C. The technique is applied to the separation of a variety of analytes which have previously been challenging or even not possible to analyze by conventional SWC. Further, the ability to use temperature and composition programming with the blended CO2/water mobile phase in SWC is also presented and discussed. Overall, the developed method considerably extends the range of non-polar analytes amenable to SWC analysis, while maintaining the beneficial conventional SWC features of flame ionization detection and environmental compatibility.

An improved interface for universal acoustic flame detection in modified supercritical fluid chromatography.

A novel method of interfacing the acoustic flame detector (AFD) with modified supercritical fluid chromatography (SFC) is presented. By applying resistive heating directly to the burner region between the restrictor outlet and the acoustic flame, infrequent severe noise, baseline drifting, and peak deformations that can occasionally be observed with the AFD are eliminated. For example, by increasing the interface temperature only a few hundred degrees Celsius, such sporadic noise in the detector can be reduced nearly ten-fold resulting in smooth stable operation of the AFD. Further, for various levels of methanol modified supercritical carbon dioxide mobile phase examined, the interface was observed to reduce detector noise in each to a common minimal range near 10-25 Hz when an appropriate temperature was achieved. The method is simply assembled, inexpensive to construct, and robust in its daily operation. Overall, the heated interface developed and presented facilitates reliable AFD operation in modified SFC, and supports further exploration and implementation of this sensor as an alternative universal detector in separations requiring an organic cosolvent in the mobile phase.