Packed Column Supercritical Fluid Chromatography Using Stainless

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Packed Column Supercritical Fluid Chromatography Using Stainless Steel Particles and Water As a Stationary Phase Jillian N. Murakami and Kevin B. Thurbide*

Downloaded by UNIV OF CAMBRIDGE on September 1, 2015 | http://pubs.acs.org Publication Date (Web): August 31, 2015 | doi: 10.1021/acs.analchem.5b02399

Department of Chemistry University of Calgary 2500 University Drive NW Calgary, Alberta T2N 1N4 Canada ABSTRACT: 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.

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supercritical CO2 as the mobile phase, whereas nonpolar analytes showed no retention. Further, since organic modifier was not used, the method was also able to employ the universal flame ionization detector (FID). Overall, while the capillary SFC-FID water stationary phase system presented beneficial properties for use in separations, certain performance attributes were noted that might be better addressed by a packed column format. For instance, good efficiency could be produced when using optimal flows with capillary columns, but separations under these conditions were very slow. Conversely, the shorter length of a typical packed column could potentially reduce such separation times at optimized flows. Further, lower polarity analytes were often little retained or separated in the capillary column system. In contrast, packed columns can potentially offer a substantial increase in the amount of stationary phase present and, hence, the retention of such analytes.21−23 In turn, this can also lead to greater column sample capacity and robustness toward the analysis of samples with particularly difficult/complex matrices.21−23 Therefore, investigating an analogous water stationary phase system in a packed column format would be useful as it could potentially expand the capabilities of this technique. The most direct approach to parallel the SS capillary SFCFID water stationary phase system in a packed column format would be to use SS particles as a packing material. However, it is uncertain if such means could support a water stationary phase for use in packed column SFC. Also, if so, it is unknown

ater has been utilized as a mobile phase component in many modes of chromatography due to its polarity, ease of use, and transparency in certain detectors. For example, highperformance liquid chromatography (HPLC),1 subcritical water chromatography,2−5 gas chromatography (GC),6,7 and supercritical fluid chromatography (SFC)8−12 have all incorporated water into the mobile phase. On the other hand, water as a stationary phase component has been relatively much less explored. For instance, early work in gas and liquid phase separations investigated aqueous additives on conventional stationary phase packings for their impact on surface activity and chromatographic selectivity.13−15 Similarly, more modern HILIC modes of HPLC also employ water/organic mobile phases to facilitate aqueous regions on hydrophilic stationary phase particles.16 Additionally, films of water on the surface of ice stationary phase particles have been used to separate analytes in HPLC as well.17,18 Recently, we introduced a novel capillary column SFC system that employs water as the stationary phase and CO2 as the mobile phase.19,20 In this system, a water stationary phase is established on the inner wall of an otherwise empty and uncoated stainless steel (SS) capillary, providing an environmentally “green” chromatographic coating that can be easily replenished. Further, while fused silica capillaries could also work in this capacity, SS capillaries were determined to be better for this purpose since they do not erode in the presence of water over the wide range of temperatures often used in SFC.19,20 In this way then, the phase was demonstrated to be stable and stationary over many different temperatures and CO2 pressures and was found to provide useful normal phase separations. Of note, polar analytes were retained quite well and produced very good peak shapes using unmodified pure © XXXX American Chemical Society

Received: June 26, 2015 Accepted: August 26, 2015

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DOI: 10.1021/acs.analchem.5b02399 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

occurred, the column outlet restrictor was connected to the detector and system operation was initiated. Chromatographic Columns. Packed columns were prepared by packing type 316 SS tubing (1/16 in. o.d. × 1 mm i.d. × 20 cm; VICI Valco) with type 316 SS powder (99.9%; −100 + 325 mesh, or about 44−149 μm diameter; Atlantic Equipment Engineers, Upper Saddle River, NJ, U.S.A.). Initially, columns were gravity packed at atmospheric pressure using sonication to help settle the particles. However, later versions were packed using water/CO2 at 360 atm to push particles into the column and reduce bed shifting (as described later in the text). Both column ends were fitted with a SS frit (0.5 μm pore size; VICI Valco) to retain the particles and a 1/ 16 in. zero dead volume SS union (VICI Valco) for connections. Under the typical operating conditions used (i.e., 40 °C and 80 atm), the pressure drop across this column was normally near 15 atm. For trials of direct comparisons with a capillary column, a 10 m length of 1/16 in. o.d. type 316 SS tubing (250 or 125 μm i.d.) was used for separations. Water Stationary Phase Collection. In order to measure the volume of the water stationary phase inside the column, the system was established as detailed above at 25 °C using 80 atm CO2. Once prepared for operation, the CO2 flow was stopped and replaced with a low pressure (10 psi) flow of N2, which was used to expel the stationary phase from the column as it was heated up to 100 °C. Meanwhile, as this water emerged from the column outlet, it was carefully collected in a dry ice-cooled vial that was capped and isolated from the minimal atmospheric moisture present. The contents of this vial were then accurately weighed, and this value was used to determine the volume of water collected. Chemicals, Reagents, and Samples. Instrument grade CO2 (99.99%; Praxair) and nitrogen-purged HPLC grade water (Honeywell Burdick & Jackson, Muskegon, MI, U.S.A.) were used. Alcohol standard stock solutions (near 20−70 μg/μL) were prepared by dissolving analytes (all ≥ 99%; Sigma-Aldrich, Oakville, ON, Canada) in HPLC grade water. For applications, eugenol (99%; Sigma-Aldrich), ibuprofen sodium salt (≥98%; Fluka, St. Louis, MO, U.S.A.), methanol (≥99.8%; EMD Chemicals, Gibbstown, NJ, U.S.A.), absolute ethanol (EMD Chemicals), and ethylene glycol (99%; Sigma-Aldrich) were used. Dried clove buds, extra strength ibuprofen liquid gel capsules (400 mg dosage), motor oil, and liqueur were all purchased from local merchants. Eugenol extractions were performed by finely grinding dried clove buds (4.0 g) and then extracting them at room temperature in ethanol (5 mL) for 28 h for complete recovery. After gravity filtration to remove the solids, the filtrate was further diluted to 10 mL in ethanol, and the solution was analyzed. For ibuprofen analyses, the contents of one liquid gel capsule were emptied into a vial and then diluted with methanol (5 mL) to reduce the viscosity prior to injection. For oil analyses, ethylene glycol was prepared (44 μg/μL) in a solution of motor oil diluted with isopropanol and hexanes (9:19:72% v/v) also to reduce the viscosity. Drinking water samples were obtained directly from local tap water and spiked (63 μg/μL) with ethylene glycol. Liqueur samples were analyzed as received. Human urine samples were deep yellow in color, as obtained from a volunteer in the early morning and spiked directly with ethanol (50 μg/μL) prior to neat injection. All other details are described in the text.

how the resulting separation properties would compare to the SS capillary SFC-FID technique. Conventionally, silica particles are most often used as packing materials in part due to their high surface area, purity, and capacity to be chemically functionalized on the surface. However, since they erode in heated water, they would not be an appropriate support here, given the wide temperature range often invoked in SFC. To the best of our knowledge, the use of only metal particles as packing materials in chromatography has not been previously reported. As such, a first step in the current context is to explore whether or not they can be used at all to support water as a stationary phase in SFC. Here, we examine the efficacy of using SS particles as a packing material to establish a water stationary phase for use in packed column SFC separations. The system is optimized and the packed column performance is directly compared with that of the SS capillary SFC-FID approach. Finally, some applications employing the packed column SFC-FID system are also examined to help demonstrate the analytical properties of this separation mode.

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EXPERIMENTAL SECTION Instrumentation and Operation. The separation apparatus employed has been detailed previously.19,20 Briefly, CO2 and water were introduced to the system using ISCO model 260D syringe pumps (Teledyne ISCO Inc., Lincoln, NE, U.S.A.). The CO2 pump was operated in constant pressure mode, where the mobile phase flow rate varied with the set system pressure. Note that all mobile phase pressures quoted herein refer to this precolumn pressure measured “upstream” at the pump. The water pump was operated in constant flow mode and was only used to prevent dehydration of the stationary phase at high temperatures (e.g., above 100 °C). Normally, at lower operating temperatures this was not necessary. Each pump outlet was connected to SS tubing (1/ 16 in. o.d. × 250 μm i.d. × 2 m; VICI Valco, Brockville, ON, Canada) that led into an HP5890A GC oven (Agilent Technologies, Mississauga, ON, Canada). These respective lines were joined inside the oven with a 1/16 in. zero dead volume SS tee union (VICI Valco). The same tubing (1.7 m) was used for CO2/water preheating and led from the tee union to the outside of the GC oven wall where it connected to a Cheminert model C4 internal sample injector fitted with a 0.5 μL sample loop (VICI Valco). For packed column trials, a short length of tubing (30 cm) connected the injector to the separation column, which was situated inside of the GC oven. For capillary column trials, the column was connected directly to the injector. The outlet of either column type then led to a linear (i.e., nontapered) deactivated fused silica capillary restrictor (Polymicro Technologies, Phoenix, AZ, U.S.A.) that was used to maintain system pressure. In normal operation this was replaced every 3 days or so. The i.d. and length of the restrictor was varied as required to control the mobile phase flow independent of pressure; however, a 15 cm length of 25 μm i.d. was commonly used. The end of the restrictor was situated inside of the FID jet about 1 cm below the burner surface. The optimal FID gas flow rates used were 260 mL/min of medical-grade air (Praxair, Calgary, AB, Canada) and 150 mL/min of hydrogen (Praxair). The detector block was held constant at 350 °C. The water stationary phase was established by pumping water through the column until it emerged at the outlet. Then the CO2 pump was set to the desired operating pressure and it displaced the excess water from the column. Once this B


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RESULTS AND DISCUSSION General Operating Characteristics. Initial results indicated that a water stationary phase could indeed be established on the SS particles and used for packed column SFC separations using a CO2 mobile phase. In fact, good peak shapes, detector stability, and separation characteristics were all noted in the early trials with SS particle packed columns. As such, it appeared that this approach could effectively promote separations of a similar nature to those seen earlier with the SS capillary SFC system.19,20 However, it became clear in these preliminary trials that the size of the SS particles initially used (nominally 44 μm diameter or less) was problematic. In particular, it was found that frequent mobile phase flow variations and blockages occurred. These issues arose from the wide particle size distribution of the SS powder employed and the presence of smaller particles in this blend that were able to escape through the column’s retaining frit and plug the flow through the restrictor. As such, a slightly larger SS particle packing material (44−149 μm in diameter) was acquired and found to provide good separations without plugging the system. Therefore, it was used exclusively in the remaining experiments. While more custom sizes and narrower distributions of SS particles would likely be further beneficial, these could not be obtained. Due to the inherent weight of the particles, a simple column packing procedure was also initially employed where the SS powder was gravity fed into the column while it was sonicated under ambient conditions. This was found to work well for several weeks, but after that time, shifting of the particle bed inside of the column was observed. In order to remedy this, the column inlet was opened and high pressure water/CO2 was used to feed additional particles into the column and stabilize the bed. This step alleviated all of the problems observed, and so it was used in all further column packing procedures. It is further interesting to note that, in addition to SS, other types of metal powders were also initially explored here as packing materials. Specifically, those available to us were a nickel, copper, and silver powder that were in the same size range (44−149 μm in diameter). In general, it was found that under the same operating conditions the SS and silver particles performed quite comparably to one another, whereas the nickel and copper powders showed very little capacity to perform separations at all. In this regard it is worth mentioning that the SS and silver particles were of “irregular” shape class, while the nickel and copper were “spherical” and this was visually confirmed with a microscope. As such, the former particles likely present a more optimal, rougher surface for water to adhere to as a stationary phase, which has been demonstrated to be very important in previous SS capillary SFC work.20 However, this requires further investigation to fully establish. Nonetheless, since nickel and copper did not perform well and because SS was more practical to use than silver, the SS particles were used in the remaining experiments. Still, a more detailed examination of other metal powders may be interesting to explore in greater detail in future studies. Optimization and Column Efficiency. With the appropriate materials and packing procedures identified, efforts were next focused on exploring the column efficiency. To optimize the mobile phase linear velocity, repeated injections of a methanol test analyte were performed at 40 °C over a range of CO2 flow rates through the column. Figure 1 presents the resulting van Deemter plot, which indicated that optimal

Figure 1. Typical van Deemter plot for different CO2 flows through the packed column using a methanol test analyte at 40 °C.

column performance was achieved using a mobile phase linear velocity near 0.4 cm/s. This value is reasonable for packed column SFC and agrees well with previous SFC results employing conventional C18 packed columns.21,22 Under these conditions, the minimum plate height for methanol was observed to be about 330 μm. Since a well packed column often displays a minimum plate height of roughly twice the average particle diameter,21,22 this value also seems practical given the SS particle size distribution used (44−149 μm diameter). Figure 2 demonstrates the column performance under these optimal conditions with the separation of a linear alcohol

Figure 2. Separation of C1−C5 n-alcohols with the packed column at 40 °C using 80 atm CO2. Elution order is 1-pentanol, 1-butanol, 1propanol, ethanol, and methanol.

mixture at 40 °C and 80 atm of CO2. As shown, the five peaks are symmetrical and well resolved. As well, they elute with the typical normal phase distribution pattern observed previously with the capillary SFC system.19,20 Also, as seen earlier,19,20 this separation behavior is primarily determined by the water solubility of the analyte. For instance, when plotting the analyte retention times of Figure 2 against their individual water solubility values, a linear relationship is established with an R2 correlation value of over 0.99. The analytes are also quite well retained in the column, with the separation completed after C


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about 160 min. These findings also reproduce well and the alcohol retention times yield RSD values of 2.6% or lower (n = 3) for repeat injections on the same column. Further, from column to column (i.e., day to day), a freshly established phase produced retention times with RSD values of about 6% (n = 3), indicating that the water also reproducibly adhered to the particles reasonably well. Since gradient programmed separations are commonly invoked in SFC to increase analysis speed, these were also investigated here. No operational problems were encountered with the packed column system as a result of programming and, as anticipated, faster analyses were obtained. For instance, Figure 3 shows a temperature-programmed separation of the

systems. Also, in comparison, the packed column was significantly more retentive than the capillary column, similar to conventional column behavior.21−23 For example, the retention factor of 1-propanol on the capillary column was near 7, while it was around 55 on the packed column. Next, in order to capture a more practical capillary column separation for comparison, an n-alcohol trial was performed using aggressive pressure and temperature gradients to elute the resolved analytes as quickly as possible. This approach is common in SFC and the typical result is shown in Figure 4. As

Figure 4. C1−C5 n-alcohol separation with a SS capillary column (10 m long × 250 μm i.d.) using a CO2 pressure program (80 atm for 70 min, 8 atm/min to 260 atm, 12 atm/min to 380 atm) and a temperature program (40 °C for 100 min, 3 °C/min to 120 °C). Elution order is 1-pentanol, 1-butanol, 1-propanol, ethanol, and methanol.

Figure 3. Temperature-programmed separation of C1−C5 n-alcohols with the packed column using 80 atm CO2. The program is 40 °C for 14 min, then 2 °C/min to 140 °C. Elution order is 1-pentanol, 1butanol, 1-propanol, ethanol, and methanol.

seen, under these more practical conditions, the capillary separation still requires about 2 h to complete, whereas the temperature-programmed packed column separation of Figure 3 is about 2.5× faster, finishing in 50 min. Once again, efficiencies are comparable between the two gradient separations, with the capillary and packed columns producing respective plate heights for 1-propanol of 120 and 105 μm. As well, the packed column remains much more retentive. For instance, even a lower polarity analyte such as 1-pentanol, which is little retained on the capillary column (retention factor near 0.1), is very well retained on the packed column by comparison (retention factor near 25). Therefore, while both the packed and the capillary column water stationary phase systems can each produce symmetrical peaks, good resolution, and comparable efficiency, the packed column provides significantly faster separations at optimal flows and greatly increased analyte retention. Water Stationary Phase Volume and Sample Capacity. The greater retentiveness of the packed column can be reasonably attributed to an increased presence of water stationary phase in the system due to the increased surface area presented by the SS particles used. In order to confirm this and better quantify the difference, experiments were performed to collect and measure the stationary phase present in the packed column. Under typical operating conditions, it was found that the packed column contains a total of about 26.3 ± 0.8 μL of water. Further, when this volume was purposely reduced under controlled dehydration conditions, analyte retention also reduced proportionately. For example, when the water content was decreased 24.6%, the retention of a

same alcohols using 80 atm of CO2. As seen, the much narrower peaks remain well resolved and separate 3× faster in under 50 min. While analyte volatility certainly plays a role in this, it should still be noted that this can also improve solubility in the supercritical CO2 mobile phase. Incidentally, the system was also compatible with the use of CO 2 pressure programming, which gave similar results. For example, at 40 °C, when increasing from 80 to 150 at 2 atm/min, the alcohols separate in about 60 min with comparable resolution and peak shape. Comparison with Capillary Column Separations. In order to better place the above packed column performance in context, comparative trials were also run with the analogous water stationary phase system employed in a SS capillary column operated under optimal flows and similar conditions. Using a typical 10 m long × 125 μm i.d. capillary column, again at 40 °C and with 80 atm of CO2, separations of the same nalcohol mixture were examined. With no gradient programming, this separation was extensive and impractically long. For instance, ethanol eluted after about 13.4 h, and methanol had still not eluted when data collection was ceased after 21 h. In contrast to this, under identical conditions, the packed column separation shown in Figure 2 demonstrates that ethanol elutes after 1.5 h, which is about an order of magnitude faster than the capillary column separation. Further, the two columns produced similar efficiency, with respective plate heights for 1-propanol of 533 and 578 μm for the packed and capillary D


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Analytical Chemistry pentanol test analyte also reduced by 21.3%. This suggests that pentanol is retained about 0.34 min/μL of water stationary phase present and indicates that retention is directly proportional to the column water content. As might be anticipated then, and similar to earlier work,19 experiments performed here in the absence of water with only bare SS particles and a CO2 mobile phase did not show any analyte retention or separation. In terms of the SS particles present in the column, the 26.3 ± 0.8 μL of water present under typical operating conditions amounts to a stationary phase load of about 5% m/m. If a median particle diameter is assumed with a spherical shape and a uniform coating, this would provide a water stationary phase film thickness of about 5 μm, which agrees with earlier estimations.19 However, this is only an approximation since more detailed information on the actual particle size distribution, shape, and coating uniformity are required to better understand this parameter. For instance, since surface roughness appears important, it could be that water preferentially coats different areas of a SS particle. Conversely, in terms of linear coverage, the stationary phase load also translates to near 131 ± 4 μL/m. This is in stark contrast to a typical SS capillary column (e.g., 250 μm i.d.), which has previously been shown to contain about 4.9 ± 0.5 μL/m of water.20 As such, this represents over 25× more water stationary phase per meter in the packed column than in a capillary column. Accordingly then, greater retention can be obtained in the shorter packed column, providing efficient and faster separations relative to the longer capillary column. Another parameter that the increased presence of the water stationary phase can directly impact is sample capacity. Of note, the packed column was found to have a much higher sample capacity than the capillary column. For example, this was investigated with injections of 1-propanol and the results indicated that the peaks yielded an asymmetry of 0.95 at 10% height for masses above 33 μg in the packed column. By comparison, the capillary water stationary phase system (e.g., 250 μm i.d.) produced the same asymmetry for 1-propanol at a nearly 20-fold lower injected mass of about 1.5 μg.20 Figure 5 further demonstrates this with the peaks arising from concentrated injections of 1-propanol in each system. As seen, at nearly 31 μg injected on the packed column, Figure 5A shows that the sample capacity has not been breached and the peak is still symmetrical. By comparison though, in Figure 5B when a much lower amount of 8.5 μg is injected onto the capillary column, the peak eluting from this long separation is severely fronted and yields an asymmetry value near 0.30. Applications. The above properties of the packed column water stationary phase SFC system can potentially provide interesting analytical utility. Figure 6 demonstrates this with the analysis of several different samples using this method. For this, elevated temperatures and pressures were also often invoked to optimize elution time. Figure 6A shows the analysis of eugenol in a clove bud extract. This analyte found in spices also has interesting medicinal qualities,24,25 and its isolation and analysis is of considerable interest.26−28 As seen, due to its relatively lower polarity and reduced water solubility, the eugenol peak (near 18 μg) is well isolated from the more retained polar ethanol solvent extract, and it readily elutes without interference in about 2 min. The results indicate a 9% m/m content of eugenol, which is typical for cloves.26,27 This is useful since the method can directly determine this analyte in such extracts, without the need for lengthy derivitization procedures that are often invoked for eugenol.28 As well, given

Figure 5. Examples of 1-propanol peak shapes for (A) 30.5 μg injected in the packed column system and (B) 8.5 μg injected in the capillary column system. Conditions are a temperature of 40 °C and a CO2 pressure of 80 atm using optimal column flows.

its short elution time, it may be of interest to explore larger bore preparative packed columns with this separation. For example, eugenol is isolated using high-speed counter-current chromatography in about 2.5 h.26 As such, the large sample capacity of the packed column water stationary phase and the inherently volatile CO2 mobile phase used here may be beneficial on a larger scale. Figure 6B shows the analysis of ibuprofen in liquid gel capsules. To do this most directly, the entire capsule contents were diluted in 5 mL of methanol (to reduce viscosity), and the sample was injected. Again, as seen, a well isolated ibuprofen peak (near 30 μg) elutes relatively quickly due to its lower polarity. Comparatively, the more polar methanol solvent and excipient matrix components (e.g., hydrophilic polyethylene glycol gellants, sorbitol, and potassium hydroxide) are strongly or even irreversibly retained in the water stationary phase. However, column fouling resulting from this is not an issue since the phase can be readily replenished. The results indicate a capsule dosage near 380 mg that aligns with the label value of 400 mg. Thus, the packed column water stationary phase SFC system can potentially facilitate determining analytes in such matrices by minimizing the sample preparation involved. This is important since sample preparation can consume near 80% of total analysis time,29 and such liquid gel formulations typically require lengthy steps of heating, filtration, dilution, and sonication to accommodate the matrix.30,31 The use of an FID here could also be beneficial if unknown degradants need to be monitored as well. Ethylene glycol is an antifreeze component that is frequently monitored since it can often contaminate engine oils and groundwater.32,33 Figure 6C shows the analysis of ethylene glycol in a motor oil sample. Since ethylene glycol is quite polar and sparingly soluble in supercritical CO2,34 a short 5 cm column was also used here to facilitate a faster elution. As seen, while the separation could still be further optimized, in this E


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In a final demonstration, this aspect is further illustrated here with the direct analysis of ethanol in different complex matrices. Figure 6E shows the analysis of ethanol in a schnapps liqueur. This aqueous sample is mainly comprised of sugar, with citric acid and flavor agents also present, and was injected neat to facilitate direct analysis. As noted in the figure, a clear ethanol peak emerges without interference from these other components since many are completely retained in the water phase and do not elute (note that a very minor peak likely due to volatile flavor additives was also detected at a retention time near 1 min before the dominant ethanol peak eluted). Incidentally, the same results were also obtained for neat coffee liqueur and mouthwash samples. In a similar but more extreme example, Figure 6F shows the analysis of ethanol in a pure untreated urine sample. Once again, despite the numerous polar matrix counterparts also present in this neat urine injection, a clear ethanol peak is obtained on a smooth baseline (note also that the retention time is slightly longer compared to Figure 6E due to a variation in the restrictor used). This can be particularly useful since the analysis of ethanol in urine is of increasing concern.35 Therefore, the high capacity of the packed column water stationary phase system can facilitate such direct analysis of challenging samples without major concern for system interference or column fouling. In this regard, it should also be noted that the water phase was quite stable when exploring these applications and produced analyte retention times with RSD values of less than 5% in each. Further, the phase did not require replenishing during the trials and was routinely used for several hours of operation/exploration at a time. Overall then, these results demonstrate that the employment of a water stationary phase in a packed column SFC format is possible through using SS particles as a packing material. This is important since it can provide a very inexpensive, simple, robust, and environmentally benign method for SFC separations of various polar analytes. Further, the method is fully compatible with the universal FID as a detector since only CO2 is used as a mobile phase and can operate in the typical temperature regime expected for conventional SFC. In the packed column format, this also has potential for scaling up to preparative quantity separations. Finally, this also opens the possibility of modifying the water stationary phase to better customize selectivity in such packed column separations toward different applications. In this way, such a technique would not be seen as a replacement to conventional GC or HPLC, but rather a complementary SFC method that could effectively handle certain separations, such as the analysis of complex aqueous samples, with relative ease and little sample preparation since the column can be readily replenished on demand. Clearly, though, in its current format, some challenges still exist for this method. For instance, the separations shown in Figures 2 and 3 are fairly long and produce quite broad peaks compared to conventional SFC methods. However, this is because the separations were carried out under optimum flow rates for comparison to the capillary separation in Figure 4 (which was even longer) and fairly mild conditions. As such, even moderately higher pressures and temperatures can greatly reduce such features. For instance, ethanol elutes 25× faster and with a much narrower peak in Figure 6E compared to Figure 2 using such an approach. More importantly, though, these observations primarily stem from the fact that quite large SS particles were employed in this

Figure 6. Analysis of various samples using the packed column system: (A) eugenol in a clove extract (40 °C and 120 atm CO2), (B) ibuprofen in a liquid gel capsule extract (50 °C and 140 atm CO2), (C) ethylene glycol in motor oil (5 cm column, 40 °C and 80 atm CO2), (D) ethylene glycol in drinking water (5 cm column, 50 °C and 90 atm (3 min) then 6 atm/min to 120 atm CO2), (E) ethanol in neat schnapps (50 °C and 140 atm CO2), and (F) ethanol in neat urine (50 °C and 140 atm CO2).

case, the numerous nonpolar oil matrix components are unretained and elute immediately, whereas the highly polar analyte is retained and elutes later. In contrast to this, Figure 6D shows the analysis of ethylene glycol in a drinking water sample. Here, a brief pressure program was used to sharpen the peak and, other than a small injection artifact observed on the short column, a clear signal arises on an otherwise undisturbed baseline. This is notable since the hardness of the local tap water used here for this sample is also fairly high at about 160 mg/L of CaCO3. Thus, despite this being coinjected with the analyte, no difficulties are noted since the salt present partitions to the water phase and remains there until the column is replenished. Therefore, not only can the water stationary phase assist in separating the analyte from the bulk of different matrix components, the high capacity of the packed column can also facilitate direct injection of such samples. Conversely, such a property also presents the possibility of adding various salts to the water phase to modify separation selectivity in future experiments. F


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study out of necessity. Of note, perhaps the biggest disadvantage to using SS particles at present is the inability to obtain smaller particles of a more defined shape and size distribution. As mentioned above, silica particles may be appropriate to use at low temperatures, but they readily begin to erode at high temperatures in the presence of water. Accordingly then, the rather large SS particle size adopted for this exploratory study also limits the plates currently realized by this method, which amount to about 2000 for the 20 cm long packed column used here. In this regard, it should also be noted that the smaller particles used initially in this study (i.e., that were discontinued due to restrictor plugging) also produced proportionately smaller plate heights and better performance before clogging the system. Therefore, the primary challenge and focus for the future development of this separation approach will be the acquisition and usage of much smaller and more conventionally sized SS particles to even further improve the efficiency and speed of this promising SFC method.

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CONCLUSIONS A novel SFC-FID system employing a CO2 mobile phase, a column packed with SS particles, and a water stationary phase was investigated. Good peak shapes and column efficiency were observed over a range of column temperatures and mobile phase pressures. The packed column water stationary phase system demonstrated faster and more retentive separations compared to analogous trials with a SS capillary column. As well, the water stationary phase volume and sample capacity were significantly greater for the packed column mode. Results show that the packed column with SS particles can provide an effective support for a water stationary phase and useful separations, particularly where analyte loads and matrix interference might be anticipated to be problematic. Future investigations with smaller particles and also more preparative scale columns may further extend the utility of this unique method.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (403) 220-5370. Fax: (403) 289-9488. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the Natural Sciences and Engineering Research Council of Canada for a Discovery Grant in support of this project.

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REFERENCES

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