Supercritical Fluid Chromatography Mobile Phase Research Articles

Development progress of stationary phase for supercritical fluid chromatography and related application in natural products

Supercritical fluid chromatography (SFC) is an environment-friendly and efficient column chromatography technology that was developed to expand the application range of high performance liquid chromatography (HPLC) using a supercritical fluid as the mobile phase. A supercritical fluid has a temperature and pressure that are above the critical values as well as relatively dynamic characteristics that are between those of a gas and liquid. Supercritical fluids combine the advantages of high solubility and diffusion, as their diffusion and viscosity coefficients are equivalent to those of a gas, while maintaining a density that is comparable with that of a liquid. Owing to the remarkable compressibility of supercritical fluids, analyte retention in SFC is significantly influenced by the density of the mobile phase. Thus, the column temperature and back pressure are crucial variables that regulate analyte retention in SFC. Increasing the back pressure can increase the density and solubility of the mobile phase, leading to reductions in retention time. The column temperature can affect selectivity and retention, and the degree to which different analytes are affected by this property varies. On the one hand, increasing the temperature reduces the density of the mobile phase, thereby extending the retention time of the analytes; on the other hand, it can also increase the energy of molecules, leading to a shorter retention time of the analyte on the stationary phase. CO2, the most widely employed supercritical fluid to date, presents moderate critical conditions and, more importantly, is miscible with a variety of polar organic solvents, including small quantities of water. In comparison with the mobile phases used in normal-phase liquid chromatography (NPLC) and reversed-phase liquid chromatography (RPLC), the mobile phase for SFC has a polarity that can be extended over a wide range on account of its extensive miscibility. The compatibility of the mobile phase determines the diversity of the stationary phase. Nearly all stationary phases for HPLC, including the nonpolar stationary phases commonly used for RPLC and the polar stationary phases commonly used for NPLC, can be applied to SFC. Because all stationary phases can use the same mobile-phase composition, chromatographic columns with completely different polarities can be employed in SFC. The selectivity of SFC has been effectively expanded, and the technique can be used for the separation of diverse analytes ranging from lipid compounds to polar compounds such as flavonoids, saponins, and peptides. The choice of stationary phase has a great impact on the separation effect of analytes in SFC. As new stationary phases for HPLC are constantly investigated, specialized stationary phases for SFC have also been continuously developed. Researchers have discovered that polar stationary phases containing nitrogen heterocycles such as 2-EP and PIC are highly suitable for SFC because they can effectively manage the peak shape of alkaline compounds and provide good selectivity in separating acidic and neutral compounds.The development of various stationary phases has promoted the applications of SFC in numerous fields such as pharmaceuticals, food production, environmental protection, and natural products. In particular, natural products have specific active skeletons, multiple active groups, and excellent biological activity; hence, these materials can provide many new opportunities for the discovery of novel drugs. According to reports, compounds related to natural products account for 80% of all commercial drugs. However, natural products are among the most challenging compounds to separate because of their complex composition and low concentration of active ingredients. Thus, superior chromatographic methods are required to enable the qualitative and quantitative analysis of natural products. Thanks to technological improvements and a good theoretical framework, the benefits of SFC are gradually becoming more apparent, and its use in separating natural products is expanding. Indeed, in the past 50 years, SFC has developed into a widely used and efficient separation technology. This article provides a brief overview of the characteristics, advantages, and development process of SFC; reviews the available SFC stationary phases and their applications in natural products over the last decade; and discusses prospects on the future development of SFC.

Supercritical fluid chromatography time-of-flight mass spectrometry enantiomeric determination of basic drugs in sewage samples

Many pharmacologically active compounds are chiral species, and their therapeutic or toxicological effects might differ between isomers. Herein, we develop a fast and sensitive chiral analysis methodology for the determination of eight pharmaceuticals, considered as emerging environmental pollutants and belonging to two different chemical classes, in wastewater and sludge samples. Compounds were separated using supercritical fluid chromatography (SFC) combined with time-of-flight mass spectrometry (TOF-MS) detection. The stationary phase, the modifier and the additive combined with supercritical carbon dioxide (CO2), in the SFC mobile phase, played a major effect in the enantiomeric resolution of selected compounds. Moreover, the composition of the mobile phase affected their ionization efficiency in the electrospray ionization source. Methanol (MeOH), containing a 0.1% of ammonia, was used as CO2 modifier for the separation of compounds in a polysaccharide-type column. Total analysis time was 15.5 min, achieving resolution factors between 1.03 and 2.49 for the eight pairs of enantiomers. In combination with mixed-mode solid-phase extraction and matrix solid-phase dispersion protocols, compounds were determined in wastewater and sludge samples, with limits of quantification in the range of 0.010–0.020 µg L−1 and 3.7–11.1 ng g−1, for aqueous and solid samples, respectively. The amine-type drugs (tramadol, propranolol and venlafaxine) were mostly found in wastewater samples, whilst azolic antimycotics were mainly quantified in sludge. The first group of compounds showed enantiomeric fractions significantly different to those existing in the commercial counterpart pharmaceuticals.

Development of a novel comprehensive analytical method for volatile compounds using supercritical fluid chromatography/mass spectrometry with a highly cross-linked styrene divinylbenzene polymer-based column.

Analytical techniques to determine volatile compounds such as flavor, aroma, and fragrances are in high demand due to their wide range of applications in industry, the chemical properties of them are very diverse. Supercritical fluid chromatography (SFC) is capable of high speed, high peak capacity separation and has a high separation coverage. It is also an advantageous for preparative purifications due to its unique mobile phase conditions. However, there is no column commercially available for SFC that is suitable to comprehensively separate volatile compounds. SFC is limited to the use of silica-based columns due to weak retentions and polymer-based column issues such as pressure, swelling and shrinkage tolerances. This study demonstrated comprehensive analytical method for volatile in SFC using a highly cross-linked styrene divinylbenzene (SDVB) polymer-based column, newly developed for SFC. In this study, 23 typical volatile compounds with a wide variety of chemical properties were selected as model compounds. The newly developed SDVB column showed, compared to conventional silica-based columns (k > 0.3), an excellent overall and substantial improved retentions (k > 1.6) under SFC mobile phase conditions. It was also able to retain esters (hydroxy acetate, pentyl butylate, methyl salicylate) and non-polar terpenes (limonene, pinene) that did not show sufficient retention in any other commercially available silica-based columns. Aldehydes reacting on NH2 column due to Schiff base formation were also successfully eluted. It was confirmed that SDVB column provided comprehensive separation and wide coverage for volatile compounds.

Replacing methanol with azeotropic ethanol as the co-solvent for improved chiral separations with supercritical fluid chromatography (SFC)

Using azeoptropic ‘190 proof’ ethanol eliminates the use of methanol in chiral super/subcritical fluid chromatography.

Effects of high concentrations of mobile phase additives on retention and separation mechanisms on a teicoplanin aglycone stationary phase in supercritical fluid chromatography

The objective of this study is to understand the behavior of a peptide in a medium containing supercritical carbon dioxide mixed with an alcohol (methanol) and acidic or basic additives in uncommonly high concentrations. Chirobiotic TAG is a chromatographic column made of silica bonded with a macrocyclic peptide, teicoplanin aglycone. With this stationary phase, two additives (trifluoroacetic acid and isopropylamine) were tested under extreme concentration conditions to observe the behavior of this peptide. Indeed, concentrations exceeding 1 M in the methanol co-solvent (>0.1 M overall concentration in the CO2-methanol mixture) were used whereas usual additive concentrations employed in supercritical fluid chromatography (SFC) rarely exceed 50 mM in the co-solvent. One purpose was to modify the apparent pH of the fluid, which is normally slightly acidic (around 5) and consequently possibly changing the ionization state of the peptide. Firstly, the effect of acidic and basic additives on the polarity and the apparent pH were evaluated with the help of color indicators. This served to assess the ionization state of the peptide under the selected operating conditions. Secondly, 54 achiral and 24 chiral molecules were injected in the chromatographic column at different levels of additives. The achiral species served at establishing retention models based on linear solvation energy relationships (LSER), while the chiral species were examined for their enantioresolution. From the LSER equations and observation of chromatograms, it appeared that specific interactions between the peptide-based stationary phase and the analytes evolved when increasing the concentration of additives, particularly hydrogen bonds and ionic interactions. A bare silica stationary phase (Acquity BEH) served as reference to deconvolute the contributions of silica support and bonded peptide. This study, with these extreme conditions of mobile phase, could be useful to understand the behavior of such peptides in SFC mobile phases and also improve the knowledge of the effects of additives in SFC, which should be helpful in the future prospect of analyzing large biomolecules in SFC.

Unexpected separate elution of cation and anion of an ammonium salt in supercritical fluid chromatography

Amines are frequently used as additives in supercritical fluid chromatography (SFC). They allow eluting basic analytes with reasonable retention times and less distorted peak shapes. Since amines are chemically active compounds, their introduction into SFC mobile phase always raises a question on whether they can react with analytes or mobile phase constituents and, if so, can it affect chromatography separation. Primary and secondary amines are known to react with carbon dioxide, also all amines, being bases, can interact with CO2-alcohol mixtures which are known to be slightly acidic. In this work, we report a case of separate elution of an ammonium salt, salbutamol sulfate, anion and cation in SFC. Retention time of a peak which molecular mass registered by mass-spectrometry in ES(-) mode corresponds to HSO4− differs substantially from the retention time of a peak corresponding to salbutamol [M+H] registered in ES(+) regime. Moreover, sulfate anion retention time depends both on amine additive type and concentration whereas salbutamol retention time doesn’t. Similar effect is observed on other columns as well as with other ammonium salts. We suppose that such behavior is caused by the exchange chemical reaction happening between ammonium salt analyte and amine additive. An additive converts a salt into a free base form and turns into a salt form itself. If this hypothesis is true, it might be important to take the possibility of such interactions into account during preparative SFC work since the compound injected might not be equivalent to a compound eluted.

Enantiomeric separation and quantification of R/S-amphetamine in urine by ultra-high performance supercritical fluid chromatography tandem mass spectrometry

To distinguish between legal and illegal consumption of amphetamine reliable analytical methods for chiral separation of the R- and S-enantiomers of amphetamine in biological specimens are required. In this regard, supercritical fluid chromatography (SFC) has several potential advantages over liquid chromatography, including rapid separation of enantiomers due to low viscosity and high diffusivity of supercritical carbon dioxide, the main component in the SFC mobile phase. A method for enantiomeric separation and quantification of R- and S-amphetamine in urine was developed and validated using ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS). Sample preparation prior to UHPSFC-MS/MS analysis was a semi-automatic solid phase extraction method. The UHPSFC-MS/MS method used a Chiralpak AD-3 column with a mobile phase consisting of CO2 and 0.2% cyclohexylamine in 2-propanol. The injection volume was 2 μL and run-time was 6 min. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions (m/z 136.1 > 119.0 and m/z 136.1 > 91.0). The calibration range was 50–10,000 ng/mL for each enantiomer. The between-assay relative standard deviations were in the range of 3.7–7.6%. Recovery was 92–93% and matrix effects ranged from 100 to 104% corrected with internal standard. After development and validation, the method has been successfully implemented in routine use at our laboratory for both separation and quantification of R/S-amphetamine, and has proved to be a reliable and useful tool for distinguishing intake of R- and S-amphetamine in authentic patient samples.

A study on the onset of turbulent conditions with supercritical fluid chromatography mobile-phases

Following a recent publication [1], the topic of turbulent flow in SFC has generated both interest and questions. Liquid-like density, coupled with significantly low viscosity of CO2-based mobile-phases may result in high Reynolds number (Re) – higher than what represents laminar flow conditions, reaching the so-called turbulent regions. Although such turbulent flows can form only in the connecting tubings, thus not directly affecting the chromatographic process, it is important to know under many situations, whether the flow inside the tubing is laminar or turbulent.In this report a comprehensive guideline to identify the possibilities of turbulent flow conditions is provided through a series of charts. Flow properties depend on state conditions (composition, pressure and temperature) and also on the tubing material and geometry. Here guidelines to detect the onset of turbulent conditions is provided for cylindrical stainless-steel tubings of different internal diameters (i.d.) under a wide range of SFC mobile-phase conditions.

Unravelling the effects of mobile phase additives in supercritical fluid chromatography. Part I: Polarity and acidity of the mobile phase

The mobile phases employed in current supercritical fluid chromatography (SFC) are usually composed of a mixture of pressurized carbon dioxide and a co-solvent. The co-solvent is most often an alcohol and may contain a third component in small proportions, called an additive (acid, base or salt). The polarity of such mobile phase compositions is here re-evaluated with a solvatochromic dye (Nile Red), particularly to assess the contribution of additives. It appears that additives, when employed in usual concentration range (0.1% or 20mM) do not modify the polarity in the immediate environment of the probe.In addition, the combination of carbon dioxide and an alcohol is known to form alkoxylcarbonic acid, supposedly conferring some acidic character to SFC mobile phases. Direct measurements of the apparent pH are impossible, but colour indicators of pH can be used to define the range of apparent pH provided by carbon dioxide-alcohol mixtures, with or without additives. Five colour indicators (Thymol Blue, Bromocresol Green, Methyl Red, Bromocresol Purple, and Bromothymol Blue) were selected to provide a wide range of aqueous pKa values (from 1.7 to 8.9). UV–vis absorption spectra measured in liquid phases of controlled pH were compared to those measured with a diode-array detector employed in SFC, with the help of chemometric methods. Based on these observations, it is concluded that the apparent pH range in carbon dioxide-methanol mobile phases is close to 5. Increasing the proportion of methanol (in the course of a gradient elution for instance) causes decreasing apparent pH. Strong acids can further decrease the apparent pH below 1.7; strong bases have little influence on the apparent pH, probably because, in this range of concentrations, they are titrated by alkoxylcarbonic acid or form ion pairs with alkoxycarbonate. However, bases and salts could stabilize the acidity in the course of gradient runs.

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control

HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π-π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical Abstract SFC-MS/MS vs. LC-MS/MS.

Comparison of liquid and supercritical fluid chromatography mobile phases for enantioselective separations on polysaccharide stationary phases

Analysis and production of enantiomerically pure compounds is a major topic of interest when active pharmaceutical ingredients are concerned. Enantioselective chromatography has become a favourite both at the analytical and preparative scales. High-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) are dominating the scene and are often seen as complementary techniques. Nowadays, for economic and ecologic reasons, SFC may be preferred over normal-phase HPLC (NPLC) as it allows significant reductions in solvent consumption. However, the transfer of NPLC methods to SFC is not always straightforward.In this study, we compare the retention of achiral molecules and separation of enantiomers under supercritical fluid (carbon dioxide with ethanol or isopropanol) and liquid normal-phase (heptane with ethanol or isopropanol) elution modes with polysaccharide stationary phases in order to explore the differences between the retention and enantioseparation properties between the two modes. Chemometric methods (namely quantitative structure-retention relationships and discriminant analysis) are employed to compare the results obtained on a large set of analytes (171 achiral probes and 97 racemates) and gain some understanding on the retention and separation mechanisms. The results indicate that, contrary to popular belief, carbon dioxide − solvent SFC mobile phases are often weaker eluents than liquid mobile phases. It appears that SFC and NPLC elution modes provide different retention mechanisms. While some enantioseparations are unaffected, facilitating the transfer between the two elution modes, other enantioseparations may be drastically different due to different types and strength of interactions contributing to enantioselectivity.

Development of an achiral supercritical fluid chromatography method with ultraviolet absorbance and mass spectrometric detection for impurity profiling of drug candidates. Part I: Optimization of mobile phase composition

Supercritical fluid chromatography (SFC) is a very useful tool in the purpose of impurity profiling of drug candidates, as an adequate selection of stationary phases can provide orthogonal separations so as to maximize the chances to see all impurities. The purpose of the present work is to develop a method for chemical purity assessment. The first part, presented here, focuses on mobile phase selection to ensure adequate elution and detection of drug-like molecules, while the second part focuses on stationary phase selection for optimal separation and orthogonality.The use of additives in the carbon dioxide – solvent mobile phase in SFC is now commonplace, and enables in particular to increase the number of eluted compounds and to improve peak shapes. The objective of this first part was to test different additives (acids, bases, salts and water) for their chromatographic performance assessed in gradient elution with a diode-array detector, but also for the mass responses obtained with a single-quadrupole mass detector, equipped with an electrospray ionization source (Waters ACQUITY QDa).In this project, we used a selection of one hundred and sixty compounds issued from Servier Research Laboratories to screen a set of columns and additives in SFC with a Waters ACQUITY UPC2 system. The selected columns were all high-performance columns (1.7–1.8μm with totally porous particles or 2.6–2.7μm with superficially porous particles) with a variety of stationary phase chemistries.Initially, eight additives dissolved in the methanol co-solvent were tested on a UPC2 ACQUITY UPC2 HSS C18 SB column. A Derringer desirability function was used to classify the additives according to selected criteria: elution capability, peak shapes, UV baseline drift, and UV and mass responses (signal-to-noise ratios). Following these tests, the two best additives (ammonium acetate and ammonium hydroxide) were tested on a larger number of columns (10) where the two additives appeared to provide very comparable overall scores. However, ammonium acetate was selected for slightly better chromatographic quality.In a second step, we investigated the effects of ammonium acetate concentration (between 0 and 25mM in the methanol co-solvent) on retention and peak efficiency. Two types of silica supports were tested by working with ACQUITY UPC2 HSS C18 SB and BEH columns. 20mM ammonium acetate in methanol with 2% water was finally selected as the best co-solvent composition.

Coupling state-of-the-art supercritical fluid chromatography and mass spectrometry: From hyphenation interface optimization to high-sensitivity analysis of pharmaceutical compounds

The recent market release of a new generation of supercritical fluid chromatography (SFC) instruments compatible with state-of-the-art columns packed with sub-2μm particles (UHPSFC) has contributed to the reemergence of interest in this technology at the analytical scale. However, to ensure performance competitiveness of this technique with modern analytical standards, a robust hyphenation of UHPSFC to mass spectrometry (MS) is mandatory. UHPSFC–MS hyphenation interface should be able to manage the compressibility of the SFC mobile phase and to preserve as much as possible the chromatographic separation integrity. Although several interfaces can be envisioned, each will have noticeable effects on chromatographic fidelity, flexibility and user-friendliness. In the present study, various interface configurations were evaluated in terms of their impact on chromatographic efficiency and MS detection sensitivity. An interface including a splitter and a make-up solvent inlet was found to be the best compromise and exhibited good detection sensitivity while maintaining more than 75% of the chromatographic efficiency. This interface was also the most versatile in terms of applicable analytical conditions. In addition, an accurate model of the fluidics behavior of this interface was created for a better understanding of the influence of chromatographic settings on its mode of operation. In the second part, the most influential experimental factors affecting MS detection sensitivity were identified and optimized using a design-of-experiment approach. The application of low capillary voltage and high desolvation temperature and drying gas flow rate were required for optimal ESI ionization and nebulization processes. The detection sensitivity achieved using the maximized UHPSFC–ESI-MS/MS conditions for a mixture of basic pharmaceutical compounds showed 4- to 10-fold improvements in peak intensity compared to the best performance achieved by UHPLC–ESI-MS/MS with the same MS detector.

Effects of mobile phase composition on retention and selectivity in achiral supercritical fluid chromatography

In supercritical fluid chromatography (SFC), the effects of adding a co-solvent to carbon dioxide are numerous. In this work, the changes in retention and selectivity occurring when the nature of the co-solvent (methanol, ethanol, isopropanol, acetonitrile) in the SFC mobile phase is varied, are studied on seven columns with different stationary phase chemistry (cyanopropyl-, pyridine-ethyl-, phenyl-oxypropyl-, phenyl-propyl-, phenyl-hexyl-, pentafluorophenyl-propyl- and octadecyl-bonded silica). Hierarchical cluster analysis and quantitative structure–retention relationships with the solvation parameter model are employed to evaluate these effects, based on the observed retention for over a hundred probe compounds. It appears that some columns are somewhat more affected than others by the change of solvent nature. Acetonitrile induces the strongest differences, essentially due to its poor capabilities as an eluent for bases and possibly because it does not adequately cover residual silanol groups. Ethanol appears to be a possible green alternative to the most-used methanol. Some hints on a possible partition–adsorption mixed mechanism are offered. Finally, sample applications with three different test mixtures relevant to cosmetic and pharmaceutical applications (flavanones, non-steroidal anti-inflammatory drugs and barbiturates) are presented.

Design and evaluation of various methods for the construction of kinetic performance limit plots for supercritical fluid chromatography

Supercritical fluid chromatography (SFC) is attributed many advantages over high performance liquid chromatography (HPLC). Next to the fact that SFC is greener than HPLC, which is especially important for preparative separations, SFC is claimed to be able to deliver faster separations at higher efficiencies (N) than HPLC. This is due to the higher diffusitivity of analytes in supercritical fluids compared to liquids (higher optimum mobile phase velocity) and to the lower viscosity of the mobile phases in SFC compared to HPLC, which results in smaller pressure drops allowing the use of longer columns and/or columns packed with smaller particles at higher velocities. In order to quantify this claimed kinetic performance advantage, it is essential to construct unbiased kinetic plots to make the comparison between HPLC and SFC. The high compressibility of the mobile phase in SFC however makes this problematic. A variable column length (L) kinetic plot method is therefore developed in this work. Because the pressure history in the column is kept constant for every data point in this method, this way of working definitely delivers exact values for the kinetic performance limits in SFC. It is shown that the traditional way of measuring the performance as a function of flow rate (fixed back pressure and column length) cannot deliver the same correct results as this variable L method. However, the isopycnic way of working on a fixed column length has also been proven to be a good alternative for the expensive and time consuming variable L method. Finally, isopycnic kinetic plots are used to compare SFC and HPLC performance in a quantitative way.

Metabolite Analysis by Supercritical Fluid Chromatography

Owing to its favorable properties, such as low viscosity and high diffusivity, a supercritical fluid can be used as the mobile phase in chromatography. Supercritical fluid chromatography (SFC) can provide high-speed and high-resolution separation. Since supercritical carbon dioxide (SCCO(2)), which is generally used as the mobile phase in SFC, is automatically emitted at room temperature, SFC is most commonly used as a preparative method. However, SFC can also be used to perform high-precision biomolecular analysis, especially for hydrophobic metabolites, because of the low polarity of SCCO(2). The use of a mass spectrometer with SFC can widen the scope of application of SFC to bioanalysis. In this review, we summarize practical application of SFC as a tool for the analysis of metabolites in real biological samples.

Supercritical Fluid Chromatographic Separation of Polyethylene Glycol Polymer

During its relativelyshort history, supercritical fluid chromatography (SFC) hasbecome an attractive alternative to GC and LC in certainindustrially important applications. SFC gives the advantageof high efficiency and allows the analysis of non-volatile orthermally labile mixtures.The density of the mobile phase in SFC is about 200-500times that in gas chromatography. The effect of shorterintermolecular distances and the resulting increase inmolecular interactions is an enhanced solubilizing capabilityof the solvent towards various solutes. Compounds withmuch higher molecular weights than gas chromatographynormally allows can therefore be chromatographed. How-ever, the most commonly used mobile phases in SFC are allrelatively non-polar fluids. Carbon dioxide, the most widelyused fluid, is no more polar than hexane,

Purification method development for chiral separation in supercritical fluid chromatography with the solubilities in supercritical fluid chromatographic mobile phases

A comprehensive approach was applied to develop a chiral purification method for an analyte that was found to be unusually difficult to scale-up in supercritical fluid chromatography (SFC). This was performed by studying major factors such as the solubility of an analyte in SFC mobile phases, impurity profiles, and cycle time. For this case study, the solubility in SFC mobile phase was measured by a packed column technique, coupled with a novel trapping mechanism to enhance measurement precision in SFC conditions. The solubility studies in SFC mobile phases suggested a couple of possible SFC mobile phases, in which the analyte would potentially be most soluble. The SFC methods were developed to purify a sample containing 15% of an impurity, after considering impurity profiles and cycle times of several potential methods in addition to SFC mobile phase solubility. An equal volume mixture of acetonitrile and ethanol was chosen for the final purification method, since this mixture demonstrated the relatively high SFC solubility among all solvent combinations with enhanced resolution between the analyte and the impurity as well as the shortest run time. The solubility of the compound was also determined in various organic solvents using a high throughput solubility screening system to better understand relative change of solubility from neat solution to SFC mobile phases.

Feasibility of Supercritical Fluid Chromatography/Mass Spectrometry of Polypeptides with Up to 40-Mers

Supercritical fluid chromatography (SFC) provides a number of advantages over traditional HPLC such as speed, practical use of longer columns, a normal-phase retention mechanism, and reduced use of organic solvents. Yet, it has been a technique traditionally limited to relatively nonpolar compounds. The nature of SFC mobile and stationary phases did not allow the elution of ionic compounds or of peptides, except, in the latter case, for the most hydrophobic peptides. The characterization of peptides is critically important for drug discovery and development in the pharmaceutical industry, as well as for a variety of other important applications. Here, for the first time to our knowledge, we show that relatively large peptides (at least 40 mers), containing a variety of acidic and basic residues, can be eluted in SFC. We used trifluoroacetic acid as additive in a CO2/methanol mobile phase to suppress deprotonation of peptide carboxylic acid groups and to protonate peptide amino groups. A 2-ethylpyridine bonded silica column, which was specifically developed for SFC, was used for the majority of this work. The relatively simple mobile phase was compatible with mass spectrometric detection.

Supercritical Fluid Chromatographic Separation of Dimethylpolysiloxane Polymer

The use of compressed (dense) gases and supercritical fluids as chromatographic mobile phases in conjunction with liquid chromatographic (LC)-type packed columns was first reported by Klesper et al. in 1962. During its relatively short history, supercritical fluid chromatography (SFC) has become an attractive alternative to GC and LC in certain industrially important applications. SFC gives the advantage of high efficiency and allows the analysis of nonvolatile or thermally labile mixtures. The density of the mobile phase in SFC is about 200-500 times that in gas chromatography. The effect of shorter intermolecular distances and the resulting increase in molecular interactions is an enhanced solubilizing capability of the solvent towards various solutes. Compounds with much higher molecular weights than gas chromatography normally allows can therefore be chromatographed. However, the most commonly used mobile phases in SFC are all relatively non-polar fluids. Carbon dioxide, the most widely used fluid, is no more polar than hexane, even at high densities. Solute polarity should be between that of the stationary phase and the mobile phase in order to have a well behaved separation. Few real samples contain only nonpolar solutes, so a major objective of research in SFC has been directed toward increasing the range of solute polarity that can be handled by the technique. To bring the SFC technique into routine use, mobile phases that are more polar than the commonly used carbon dioxide are necessary. The solvent strength of supercritical CO2, even at high density, is not sufficient for the elution of polar solutes. Polar mobile phase such as NH3 exhibit useful properties, but a more practical way to extend the range of compounds separable by SFC is to use a mixed mobile phase. The solubility of the solute in the supercritical phase can be influenced considerably by adding modifiers to the mobile phase. The use of modifiers has been reported by Jentoft and Gouw and by Novotny et al. The latter group showed that adding 0.1% 2-propanol to pentane as the mobile phase decreases the observed partition coefficient (K) values for many polynuclear aromatic hydrocarbons by 20-35%. Thus, the addition of modifiers (generally organic solvents) to a supercritical mobile phase changes the polarity of the mobile phase and also leads to deactivation of the column packing material. In capillary SFC, most separations are made with pure CO2 because of its compatibility with an flame ionization detection (FID); except for formic acid and water, the addition of any common modifier precludes the use of FID. Modifiers are essential in packed-column SFC for the elution of polar compounds and are extensively used. Several workers have reported the influence of modifiers on peak shape, selectivity and retention time in capillary and packed-column SFC. A simple and effective way to add modifiers to a supercritical fluid mobile phase is to use a saturator column, which is usually a silica column saturated with polar alcohols. In this work, water was used as a polar modifier and a μporasil column as a saturator column. The μ-porasil column was inserted between the pump outlet and the injection valve. During the passage of the supercritical fluid mobile phase through the silica column, a polar modifier (water) can be dissolved in the pressurized supercritical fluid. Dimethylpolysiloxane polymer has been known as more polar polymer than polystyrene polymer. Dimethylpolysiloxane polymer has never been separated using water modified mobile phase. In this paper, using a μ-porasil column as a saturator column, excellent supercritical fluid chromatograms of dimethylpolysiloxane oligomers were obtained.