Considerations on the possibilities and limitations of comprehensive normal phase-reversed phase liquid chromatography (NPLC x RPLC)

A comprehensive normal phase system LC-reversed phase LC (NPLC x RPLC) was evaluated for the separation of a pharmaceutical mixture and citrus oil extracts. NPLC was performed on a 25 cm x 1 mm ID x 5 microm dp diol phase. In the second dimension, an RP 18 monolithic column (10 cm L x 4.6 mm ID x 2 microm macropore size) and an octadecyl silicagel-packed column (5 cm L x 4.6 mm ID x 3.5 microm dp) were applied for the analyses of the pharmaceutical sample and the citrus oil extracts, respectively. A two-position/ten-port switching valve was used as interface. Under optimised LC conditions, the high degree of orthogonality between NP and RP resulted in peak capacities of 300 for the pharmaceutical sample and of 450 for the citrus oil extract composed of lemon and orange oil. Despite the features of NPLC x RPLC, several shortcomings related with the solvent incompatibility between the two LC modes were identified and the practical consequences were discussed.     

An attempt to extend ordinary capillary gas chromatography to supercritical fluid chromatography (SFC)

Since capillary columns with well immobilized stationary phases are expected to withstand contact with supercritical fluids, we wished to study their amenability to SFC. Simultaneously, we wished to learn how far SFC can be accomplished with the ordianary tools of capillary GC. The study demonstrates that truly supercritical, not just relatively high, pressure is required to ensure the typical effects of SFC. Results obtained with sub-and supercritical pressure are compared and discussed. A comprehenshive study of the parameters permitting SFC with capillary GC equipment showed a clear preference for CO₂ as a carrier, FID detection, and oncolumn sampling. While no additional equipment is required, a critical feature is the flow restrictor to be mounted on the end of the column. The production and properties of this restrictor are discussed in detail. It is reasonable to hope that SFC with 0.1 mm id capillary columns can be realized in the pressure range of 100–150 bar, where substances which cannot be eluted from a capillary colum under GC donditions are expected to be analyzed.     

Rapid and high resolution capillary supercritical fluid chromatography (SFC) and SFC/MS of trichothecene mycotoxins

The potential application of capillary column supercritical fluid chromatography (SFC) and SFC/mass spectrometry (SFC/MS) for the separation and analysis of mycotoxins of the trichothecene group was examined. Trichothecenes present significant analytical problems for both gas and liquid chromatography with a major difficulty for the latter being the lack of sufficiently sensitive and selective detectors. Supercritical carbon dioxide mobile phases at temperatures up to 100 degrees C were used with deactivated fused silica columns coated with crosslinked stationary phases. Separations were obtained under pressure ramped conditions using long (15 m) 50-micron i.d. columns for several trichothecenes (diacetoxyscirpenol, deoxynivalenol, and T-2 toxin) and related higher molecular weight macrocyclic (roridin and verrucarin) trichothecenes. In addition, new rapid pressure programming techniques with short (less than 2m) 25- to 50-micron i.d. capillary columns were used to obtain fast separations in as little as 1 min. SFC/MS with ammonia chemical ionization provided high selectivity and sensitive detection (with approximately 1-pg detection limits) for trichothecene mixtures. The extension to complex sample matrices is discussed and the application of selective MS/MS detection is demonstrated.     

Capacity ratios and diffusion coefficients of low-volatile organic compounds in supercritical carbon dioxide from supercritical fluid chromatography (SFC)

Capacity ratios k have been determined for phenyl myristate, phenyl palmitate and phenyl stearate from supercritical fluid chromatography (SFC) at 35 to 56°C and 85 to 190 bar using supercritical CO₂ as mobile phase and Perisorb A and Perisorb RP8 as stationary phases. For the esters used a rise in pressure from 90 to 190 bar produces a drop of k by about two orders of magnitude giving evidence of the rapidly increasing solvent power of supercritical CO₂ with increasing density. At a constant CO₂ density, k decreases with increasing temperature. The separation of the esters was found to be the better the lower the pressure was.

An apparatus for the chromatographic determination of binary diffusion coefficients in supercritical gases is described. D₁₂ values for some organic compounds in carbon dioxide at 40°C and in the pressure range from 80 to 160 bar are presented. With an improved electronic flow regulator an overall precision of the D₁₂ values of ±3% is obtained.     

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.     

A new interface for coupling solid phase microextraction with liquid chromatography

A modified Rheodyne 7520 microsample injector was used as a new solid phase microextraction (SPME)–liquid chromatography (LC) interface. The modification was focused on the construction of a new sample rotor, which was built by gluing two sample rotors together. The new sample rotor was further reinforced with 3 pieces of stainless steel tubing. The enlarged central flow passage in the new sample rotor was used as a desorption chamber. SPME fiber desorption occurred in static mode. But all desorption solvent in the desorption chamber was injected into LC system with the interface. The analytical performance of the interface was evaluated by SPME–LC analysis of PAHs in water. At least 90% polycyclic aromatic hydrocarbons (PAHs) were desorbed from a polyacrylonitrile (PAN)/C18 bonded fuse silica fiber in 30 s. And injection was completed in 20 s. About 10–20% total carryovers were found on the fiber and in the interface. The carryover in the interface was eliminated by flushing the desorption chamber with acetonitrile at 1 mL min⁻¹ for 2 min. The repeatability of the method was from 2% to 8%. The limit of detection (LOD) was in the mid pg mL⁻¹ range. The linear ranges were from 0.1 to 100 ng mL⁻¹. The new SPME–LC interface was reliable for coupling SPME with LC for both qualitative and quantitative analysis.     

Integration of normal phase liquid chromatography with supercritical fluid chromatography for analysis of fruiting bodies of Ganoderma lucidum

In this study, a comprehensive 2-D chromatography was constructed, consisting of normal phase LC (NPLC) with a CN column as the first dimension, and supercritical fluid chromatography (SFC), with a Merck Chromolith Flash C(18) column as the second dimension, which were connected by a 10-port, dual-position valve controlled automatically by a self-designed software. Such platform was applied into the analysis of the fruiting bodies of Ganoderma lucidum, a traditional Chinese medicine, and within 2?h analysis, the obtained peak capacity of the 2-D-NPLC-SFC system was about 350, obviously higher than that of each dimension. These results demonstrate that 2-D-NPLC-SFC is not only of good orthogonality, but also of high throughput for the analysis of complex samples.     

Effect of temperature in reversed phase liquid chromatography

The high temperature liquid chromatography (HTLC) reveals interesting chromatographic properties but even now, it misses some theoretical aspects concerning the influence of high temperature on thermodynamic and kinetic aspects of chromatography: such a knowledge is very essential for method development. In this work, the effect of temperature on solute behavior has been studied using various stationary phases which are representative of the available thermally stable materials present on the market. The thermodynamic properties were evaluated by using different mobile phases: acetonitrile-water, methanol-water and pure water. The obtained results were discussed on the basis of both type of mobile phases and type of stationary phases. Type of mobile phase was found to play an important role on the retention of solutes. The kinetic aspect was studied at various temperatures ranging from ambient temperature to high temperature (typically from about 30 to 200 degrees C) by fitting the experimental data with the Knox equation and it was shown that the efficiency is improved significantly when the temperature is increased. In this paper, we also discussed the problem of temperature control for thermostating columns which may represent a significant source of peak broadening: by taking into account the three main parameters such as heat transfer, pressure drop and band broadening resulting from the preheating tube, suitable rules are set up for a judicious choice of the column internal diameter.     

Construction of on-line supercritical fluid extraction with reverse phase liquid chromatography–tandem mass spectrometry for the determination of capsaicin

A column-switching system, composed of supercritical fluid extraction (SFE) and reverse phase liquid chromatography/mass spectrometry (RPLC/MS) was constructed for on-line extraction and reverse-phase separation of capsaicinoids in capsicum fruits.     

Coupling miniaturized liquid chromatography to capillary gas chromatography (micro-LC-CGC): Possibilities of reversed phase LC

Retention gaps with different polarity treatments were evaluated for reversed phase solvents. Aminopropyl- and cyanopropyl-deactivated retention gaps showed the best results for methanol-water mixtures. A reversed phase packed fused silica capillary LC column is connected on-line with a capillary gas chromatography column. The combination was used for the analysis of diazepam in urine. Volume overloading on packed fused silica columns without loss of too much efficiency was demonstrated for propranolol.     

Selectivity changes towards steroids in packed column supercritical fluid chromatography (SFC) induced by temperature and pressure variation

Summary Pressure/temperature variation in SFC was shown to influence selectivity towards steroids. Major changes were observed for polar column packings compared to non-polar packings and variation of the amount of modifier showed that this is valid over the whole investigated range. The effect is underlined by a fall in resolution in the low density range (low pressure/high temperature) for polar stationary phases and appears to be significantly larger than for non-polar phases. Major selectivity shifts induced by variation of the physical properties of the mobile phase are considered to be due to the greater effect of polar modifiers on the nature of polar stationary phases than on non-polar phases.