Effect of stationary phase film thickness on efficiency in capillary supercritical fluid chromatography

Fused-silica capillary columns with internal diameters of 50 μm were coated with 0.25 to 1.0 μm films of SE-54 and evaluated under supercritical fluid chromatographic conditions using carbon dioxide as mobile phase. Experimental results compared well with theoretical predictions. There was no significant difference in h_min or ū_opt for film thicknesses from 0.25 to 1.0 μm over k = 1 to 5. At a film thickness of 1.0 μm, calculations indicate that approximately 10% resolution loss would be expected for solutes with k = 1.     

Introduction of large sample volumes in capillary supercritical fluid chromatography

A splitless injection method using make-up flow was developed for SFC. Dilution of sample solvent with carbon dioxide mobile phase was very effective for focusing the solutes onto the column. Injection of a 4.5-μl sample volume on a 100-μm i.d. capillary column became possible.     

Analysis of commercial dyes by capillary column supercritical fluid chromatography

High resolution separations of selected commercial azo, aniline, and anthraquinone dyes by capillary column supercritical fluid chromatography (SFC) are demonstrated. Supercritical n-pentane was used as a mobile phase and provided efficient separations of multi-functional, polar disperse dyes with molecular weights up to approximately 700 daltons.     

Retention in capillary supercritical fluid chromatography

Supercritical fluid chromatography (SFC) sometimes exhibits GC-like behavior and sometimes LC-like behavior, depending on conditions. However, it is not always clear whether one of these types of behavior, or a combination, operates for a particular set of conditions for every solute in a mixture. For example, some components may be partitioned mostly by their vapor pressures, while others, in the same mixture, are partitioned predominantly by solvent-like properties of the mobile phase. Plots of retetion (as log of the capacity factor) vs. reciprocal temperature at constant pressure reveal a clear change in the character of the separation of well-behaved solutes. A thermodynamic explanation of the observed behavior is given, based on the assumption that partitioning is controlled by the heats of solution of solute in the mobile and stationary phases. A model of SFC retention as it deviates from pure-GC behavior on the same column is presented.     

Current technological challenges in capillary supercritical fluid chromatography

In cases where high efficiency is required to resolve complex mixtures of either thermally labile or nonvolatile organic compounds, capillary supercritical fluid chromatography may be the most desirable analytical method. While great strides in this new technology have been made over the last few years, several problem areas are requiring increased attention. These include sample introduction systems, pressure reduction at the end of the column, column stability in various supercritical mobile phases, and migration of polar solute molecules. This paper describes the state-of-the-art in capillary SFC with emphasis on the progress made and future needs in the solutions to these specific problems.     

Splitless injection in capillary supercritical fluid chromatography

A splitless injection technique, allowing 0.5 μl injections on 50 μm i.d. columns, has been developed.     

Serially coupled capillary columns supercritical fluid chromatography with midpoint pressure control

Two capillary columns of different polarities were coupled in series by means of a coupling restrictor. The pressure of the first column and the midpoint pressure (between the coupling restrictor and the second column) were controlled independently of each other using two pumps. The selectivity of this separation system was highly dependent on the pressure difference and could be continuously changed between those of two columns. The pressure difference could be changed even in course of separation for fine tuning of the selectivity. Several examples were shown to demonstrate the utility of this method.     

Open tubular column supercritical fluid chromatography/mass spectrometry on a benchtop mass spectrometer

A benchtop SFC/MS system is described which utilizes supercritical carbon dioxide in a 50 micron diameter open tubular column interfaced directly to an unchanged commercially available benchtop capillary GC/MS system equipped with a chemical ionization (CI) source. A small amount of methane reagent gas was admitted co-axially to a capillary restrictor at the exit of the capillary chromatographic column. This make-up gas served as the CI reagent gas and appeared to optimize the sensitivity of the system while providing abundant (M+1) ions for the analytes investigated in this study. Good chromatographic intergrity was obtained for the GC/MS test compound, decafluorotriphenylphosphine (DFTPP), but the capillary restrictor appeared to cause some tailing of the ion current profiles resulting from low nanogram levels of caffeine and some fatty acid esters. Improvements in the SFC/MS capillary restrictor interface and the pumping system of the benchtop GC/MS system should increase the capability of this system for future applications.     

Temperature programming elution in capillary supercritical fluid chromatography

Summary Effects of column temperature on the retention behaviour of aromatic hydrocarbons and dialkyl phthalates were investigated in capillary supercritical fluid chromatography (SFC) with carbon dioxide as the mobile phase. Negative temperature programming could partly replace pressure programming. Positive temperature programming was applicable to solutes with proper volatility, in which gas chromatography-like retention mechanism (partition process) was involved.     

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.     

The effects of the column pressure drop on retention and efficiency in packed and open tubular supercritical fluid chromatography

The effects of the pressure drop across the column on retention and efficiency in SFC have been studied. Numerical methods are described which enable the prediction of hold-up time and pressure drop in both packed and open tubular columns. Predictions of both hold-up time and pressure drop are in good agreement with experimental data. The density gradient along the column can be calculated using the numerical methods and a procedure is described which enables the calculation of the overall capacity factors of the solutes from the density profile in the column. Significant variations of the capacity factor are observed along the column. The effect of the density gradient along the column on local diffusivity and dispersion is studied. The column efficiency in systems with significant pressure drops is affected by changes in: the linear velocity of the mobile phase; the diffusion coefficients; and the capacity factors of the solutes along the column. The overall efficiency of the chromatographic system can be calculated if, as is the case for open tubular columns, adequate plate height equations are available.     

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.     

Direct coupling of capillary supercritical fluid chromatography with double-focusing high resolution mass spectrometry

An integral restrictor interface with jet separator for coupling capillary column supercritical fluid extraction – supercritical fluid chromatography with high resolution mass spectrometry (SFE-SFC-MS) has been built and used for the analysis of a fatty acid ester, and of polymer additives with a wide range of masses. The mobile phase used was supercritical carbon dioxide; a flame ionization detector (FID) was used in parallel with the mass spectrometer. Different SFC-MS interface operating conditions, e.g. temperature, restrictor position, flow rate, and sample transfer conditions were optimized to obtain good sensitivity and separation for these applications. In addition, the sensitivity of measurements performed with the direct insertion probe and by SFC-MS interface have been compared.     

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.     

Low flow rate modifier addition in packed capillary column supercritical fluid chromatography

A new method to accurately deliver small amounts (0.5 to 20 mol%) of modifier into CO₂ was used to study the effects of three different modifiers (methanol, water, and formic acid) in packed capillary column SFC. The method allows the use of different modifiers, with minimal instrument modification. The effects of the different modifiers at different concentrations on retention and peak shape are shown by analyzing a polarity test mixture and a sample of free fatty acids.