New device for controlling the amount of methanol in carbon dioxide mobile phase for supercritical fluid chromatography

A new device to accurately deliver a small amount of methanol into supercritical carbon dioxide fluid is described. Carbon dioxide, the most widely used mobile phase in supercritical fluid chromatography, is a relatively non-polar fluid, and hence the addition of a small amount of methanol could change the solvent strength of the mobile phase. In this work, supercritical CO₂ and methanol are delivered from the pump to a 100-μl mixing chamber in which a small magnetic bar is rotating. After passing through the mixing chamber, supercritical CO₂ is changed to a new mobile phase with different polarity. The modified mobile phase was successfully used for the separations of polar compounds and polyaromatic hydrocarbons (PAHs).     

Polyacrylate liquid crystalline stationary phases in supercritical fluid chromatography with carbon dioxide mobile phase

Summary New polyacrylate liquid crystalline compounds were coated onto glass or fused-silica capillary columns as stationary phases and applied to supercritical fluid chromatography. These stationary phases, were very stable: no bleeding was observed at 200°C and up to 200kg/cm² pressures of carbon dioxide mobile phase. The wide working range of the capillary column was extended below the g-n transition temperature. Isomeric compounds such as - and -methoxynaphthalene, anthracene and phenanthrene and several phenolic compounds were separated.     

Sulfur hexafluoride as a mobile phase for supercritical fluid chromatography

Summary The capabilities of sulfur hexafluoride as a mobile phase for supercritical-fluid chromatography are investigated. An evaluation of its overall utility on the basis of separations of standard aromatic hydrocarbon odel mixtures performed on a variety of bonded-phase, packed columns with UV detection is presented. The dependence of separation performance upon operational parameters is also examined. A comparative evaluation of the chroamtographic properties of supercitrical sulfur hexafluoride and those of supercritical carbon dioxide is developed from these separations under corresponding supercritical state conditions.     

Efficiency losses caused by gradients in solute velocity in packed column supercritical fluid chromatography

Summary Under a few sets of extreme conditions, retention gradients caused losses in apparent efficiency in packed column SFC. Under most practical chromatographic conditions no such losses occurred. The efficiency of five columns were measured both individually and connected in series. Under most conditions, no loss occurred. Under a few sets of conditions, the apparent efficiency of the five columns connected in series was as much as 66 % lower than the sum of the efficiencies of the individual columns measured under the same conditions. To observe a loss, the local retention near the column outlet must be unrealistically high and a steep non-linear change in retention is required. Under some sets of conditions, an approximate form of a recently proposed theory predicted losses similar to those observed.     

Computer-assisted retention prediction of polycyclic aromatic hydrocarbons in supercritical fluid chromatography using carbon dioxide as the mobile phase

Summary The dependence of the capacity factor of polycyclic aromatic hydrocarbons on column temperature and on the density of the mobile phase in supercritical-fluid chromatography was investigated using carbon dioxide as the mobile phase. Logarithmic capacity factors of polycyclic aromatic hydrocarbons were obtained as a linear function of the reciprocal column temperature at a constant molar volume of carbon dioxide.The application of the Retention Prediction System to supercritical-fluid chromatography is demonstrated: one can predict the retention of polycyclic aromatic hydrocarbons using equations including column temperature, density and the physico-chemical properties of the solutes as the parameters.     

Effect of column diameter on efficiency in capillary supercritical fluid chromatography

Fused silica capillary columns with internal diameters from 100 to 25 μm were coated with SE-54 and evaluated under supercritical fluid chromatographic conditions using carbon dioxide as mobile phase. Experimental results compared well with theoretical predictions. At ten times the optimum mobile phase velocity and for a capacity factor, k of 3, efficiencies of 2300 to 5600 plates m^?1 were obtained for column diameters of 100 to 25 μm, respectively.     

Capillary supercritical fluid chromatography of limonoids

Capillary supercritical fluid chromatography (SFC) with carbon dioxide as the mobile phase has been used to separate a mixture of limonoid standards. Chromatographic conditions were optimized to enable analysis of plant extracts. The seed of Aphanamixis polystacha (Meliaceae) and the stem bark of Harpephyllum caffrum (Anacardiaceae) were extracted with hexane and the extracts were analyzed by SFC to demonstrate the potential of the technique as a screening tool. SFC analysis indicated the presence of limonoids in the hexane extract of the bark of Entandrophragma delevoyii (Meliaceae). One limonoid and two protolimonoids isolated from the extract were separated by SFC.     

Effect of mobile phase density gradients on efficiency in packed column supercritical fluid chromatography

Summary The efficiency of packed columns was measured as a function of flow rate, temperature, outlet density, and the density differential across the column, unsing pure carbon dioxide as the mobile phase. Although density differentials are often blamed for a serious loss in efficiency in packed column supercritical fluid chromatography, the results show that efficiency was not a function of the density differential. Peak shapes suggest that apparent loss in efficiency is actually due to inadequate solubility of the solute in carbon dioxide.     

On-line multidimensional supercritical fluid chromatography/capillary gas chromatography

A new analytical technique combining on-line supercritical fluid chromatography with capillary gas chromatography has been developed. The supercritical fluid sample effluent is decompressed through a restrictor directly into a conventional capillary gas chromatographic injection port. This technique allows for not only direct (100%) sample transfer from the supercritical fluid chromatograph to the gas chromatograph but also for selective or multi-step heartcutting of various sample peaks as they elute from the supercritical fluid chromatograph. Heartcut times are determined by monitoring the responses from the flame ionization or ultraviolet absorbance detectors on the supercritical fluid chromatograph. This report describes the operational setup and provides the results of heartcut reproducibility experiments using normal hydrocarbon and aromatic test mixtures. Results from studies where operational parameters were varied, such as GC injector temperature, will also be provided. The potential usefulness of this new technique for selective heartcutting will also be demonstrated using complex hydrocarbon streams.     

Fundamental challenges and opportunities for preparative supercritical fluid chromatography

The use of supercritical fluids as mobile phases in chromatography was suggested nearly fifty years ago. In spite of some major potential advantages, this mode of chromatography, generally known as SFC, is only now beginning to be considered by the mainstream community but it still does not yet enjoy a popularity comparable to those of gas or liquid chromatography. This seems to be largely due to a combination of (1) the serious instrumental difficulties that took many years to solve; (2) the complexity of the behavior of supercritical fluids in chromatographic systems when their temperature, pressure, or composition changes; (3) the long-lasting absence of any substantial incentive to use more complex systems, when the simpler and more robust approaches provided by HPLC are available. This situation, however, has begun to significantly change during recent years. The incentive of employing green, sustainable technologies in industrial processes as well as in analyses is increasing. Because mobile phases generally used in SFC tend to be less environmentally harmful and less expensive than those used in HPLC, SFC presents strong economical and regulatory advantages over the latter technique. Added to that, steady advancements in LC techniques in the last three decades has solved many instrumental difficulties related to SFC, which is now taking full advantages of many of these advances. One factor, however, has remained mostly unresolved. A clearer understanding of the physico-chemical behavior of supercritical fluids in preparative chromatographic columns under nonlinear conditions is still needed. This seems to be the main obstacle to the establishment of SFC as a sustainable separation tool. One aim of this review is to highlight these issues in more detail through a survey of the state-of-the-art techniques available for the design and operation of SFC. Another aim is to outline a possible series of investigations, which are necessary to develop a better physical understanding of SFC.     

Retention behaviour in liquid and supercritical fluid chromatography using methanol or diethyl ether as mobile phase

Summary Effects of column temperature and pressure on the retention behaviour of aromatic hydrocarbons were investigated in liquid and supercritical fluid chromatography by using a packed column and methanol or diethyl ether as the mobile phase. Oligomers of styrene, polysiloxane and a non-ionic detergent were separated by temperature- and pressure-gradient elution.     

Packed column supercritical fluid chromatography of isomeric polypeptide pairs

The characterization and determination of peptides is of great importance in the pharmaceutical industry as is the ability to rapidly perform targeted determinations of bioactive peptides in complex matrices. The purpose of the presented work is to assess the feasibility of packed column supercritical fluid chromatography (SFC) for the separation of two-pairs of water soluble peptides of identical mass, composition and charge that differ only in amino acid sequence. Upon evaluating a variety of conditions, trifluoroacetic acid (HTFA) in conjunction with methanol as the modifier proved to be, in general, the most successful mobile phase additive for elution of the two isomeric peptide pairs from all nitrogenous stationary phases. In contrast, water and ammonium acetate gave distorted peak shapes and therefore proved to be less satisfactory as neutral additives. The basic additive, iso-propylamine (IPAm), coupled with HA-Pyridine yielded the highest resolution factor for the complete study. Aminopropyl and HA-Pyridine columns with 5 μm particle size and 60 Å pore size were found to be best for resolution of each peptide pair. Bare silica and phenyl-hexyl stationary phases did not afford any separation. The primary roles of the carbon dioxide and methanol modifier are believed to provide (a) stationary phase solvation and (b) peptide solubility and transport; while, HTFA is postulated to fully protonate each peptide and form ion pairs between its conjugate base and cationic peptide analyte. The separation process, therefore, is best viewed as ion pair supercritical fluid chromatography (IP-SFC). For the case where IPAm gave good resolution on the HA-Pyridine column, the peptides are probably in the neutral state.     

Direct on-line coupling of small subcritical and supercritical fluid extractors with packed column supercritical fluid chromatography

Summary A mini extractor of 85 L void volume and a micro extractor of 3–4 L void volume have been coupled directly with a packed column SFC and used under sub- and supercritical conditions. The mini extractor is suitable for holding adsorbates which can be on-line extracted and the extract chromatographed (direct SFE-SFC). The micro extractor can be used for direct sample introduction of liquid and solid materials under SF conditions. Thus any solvent interference with the sample and the chromatographic conditions is excluded. Standard samples of wood tar residue, engine oil, and metal organic compounds have been tested.     

Optimization by isochronal analysis I. Changes in mobile phase composition and velocity

Summary The theory of isochronal analysis (time normalization) has been extended to reversed-phase liquid chromatography. Of the possible manipulation of experimental parameters, the present paper develops a framework for optimizing the resolution by the simultaneous change of mobile phase velocity and composition. For that purpose the resolution equation was written in terms of these two parameters and under the constraint of constant analysis time. This treatment allows plotting the resolution versus the mobile phase composition and velocity. The surfaces thus obtained show the changes that the analyst must perform in order to optimize the resolution. The treatment deals with three possible α dependencies on the amount of organic modifier: constant, increasing and decreasing. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

The dependence of reduced plate height on reduced velocity in carbon dioxide supercritical fluid chromatography with packed columns

Summary The relation is proposed for the dependence of reduced plate height, h, on reduced velocity in carbon dioxide, supercritical fluid chromatography with packed columns. The classical Knox model does not take into account the density drop in the column which produces a peak-broadening. A supplementary coefficient with a parabolic dependence on velocity must be added and then a good fit between experimental results and theory is observed. Finally, the influence of other parameters such as pressure, capacity factor and solute molecular area is discussed.     

Bioanalytical application of multidimensional open tubular column supercritical fluid chromatography

Summary On-line multidimensional open tubular column supercritical fluid chromatography (SFC/SFC) using either a flow-switching or a rotary valve-switching interface has been applied to bioanalytical problems. These include the analysis of (a) cholesterol in dried egg yolk, (b) retinoic acids in rat serum, and (c) a digitalis-like factor in peritoneal dialysate from hypertensive patients. A solvent vent injection technique was incorporated in the system, allowing single or multiple volumes of extract (up to 2.0 L each) to be injected into an uncoated, but deactivated, length of capillary precolumn without flooding of the analytical column. For flow-switching, a well-deactivated, glass-lined offset-cross with a small dead volume was placed between the primary and the secondary column. With a rotary valve-switching interface, a cold trap was employed for refocusing analytes at low pressure from single or multiple fractional cuts after being transferred to the second dimension.     

Fast supercritical fluid chromatography of polymers using packed capillary columns

Summary Fast separations of perfluorinated polyethers and polymethylsiloxanes that are composed of 50–80 oligomers were demonstrated in packed capillary column supercritical fluid chromatography (SFC) using a carbon dioxide mobile phase. Separations were accomplished within 10 min using a 13 cm×250 μm i.d. column packed with 2 μm porous octadecyl bonded silica (ODS) particles. Effects of particle diameter of the packing material and pressure programming on separation were investigated, and packed column SFC was compared with open tubular column SFC. Results show that as the particle diameter was decreased from 5 to 3 to 2 μm and the column length was reduced from 85 to 43 to 13 cm, the separation time could be reduced from 70 to 20 to 10 min while still maintaining similar separation (resolution). Short columns packed with small porous particles are very suitable for fast SFC separations of polymers.     

Investigation of peak compression effects in packed column supercritical fluid chromatography using chemometrics

Summary In this paper chemometrics have been used to study and characterize peak compression phenomena in packed column SFC. A carbon dioxide/2-methyl-1-propanol mobile phase was used in the experimental design (modifier concentration, temperature and pressure) and modelling part of the investigation. A cubic interaction term was needed in the model to obtain a reasonable fit, suggesting that all three parameters are of significance in terms of controlling peak compression. At the optimum conditions derived from the model a narrow peak was obtained as predicted.