Design of a prolog-based expert system for planning separations of steroids by high-performance liquid chromatography

A Micro-PROLOG-based expert system is described for planning separations by high-performance liquid chromatography. The criteria for the system design are presented together with details of the PROLOG implementation and its application to the separation of steroids.     

Separation of urinary ultraviolet-absorbing metabolites by high-pressure liquid chromatography using a commercially available analytical unit

Two biphasic systems, both consisting of water, dextran and poly(ethylene glycol) but differing in the molecular weight of the latter polymer, Mr = 35 000 (systems A) and 6000 (systems B), have been studied. The phase diagrams for the two systems at 20 degrees are compared. The partition of proteins influenced by salts and the viscosity of the phases are direct functions of the difference in composition between the upper and the lower phase as measured by the lengths of the tie-lines found for the systems in the phase diagram. The solubility of gamma-globulin is 1.5-2 times larger in an A system than in a B system with corresponding tie-line. In both systems, poly(ethylene glycol) palmitate has less effect on the partition of serum albumin than would be predicted from the distribution of poly(ethylene glycol) and reflects the non-ideality of simple equilibrium models.     

Sub-second liquid chromatographic separations by means of shear-driven chromatography

Utilizing the concept of shear-driven chromatography, we have been able to realize reversed-phase LC separations in flat rectangular nano-channels coated with a C8 monolayer and being as thin as 100 nm. At this scale, the separation kinetics are strongly enhanced, as is witnessed by the extremely short time (< 0.1 s) needed to separate a mixture of coumarin dyes. The observed plate numbers are still relatively small, because the experiments were conducted in ultra-short columns (< or = 1 mm) and under injection band width-limiting conditions.     

Separation of nitroimidazoles by reversed-phase high-pressure liquid chromatography

A mixture of eight N-substituted and unsubstituted nitroimidazoles has been separated by high-pressure liquid chromatography with 5% ethanol as the eluent. Compounds with the same capacity ratios were selectively detected electrochemically by differential pulse polarography with a hanging mercury drop electrode. The HMDE detector had higher detection limits than the photometric detector set at 315 nm.     

Semi-xylenol orange and its purification by high-pressure liquid chromatography

By Mannich condensation of o-Cresol Red, iminodiacetic acid and formaldehyde, Semi-Xylenol Orange (SXO) has been prepared in a 10-hr batch-procedure with a yield of about 30%. From the crude product SXO has been isolated by reversed-phase HPLC with perchloric acid-acetone mixtures as the mobile phase and C(18)-bonded silica as the stationary phase. The SXO fraction was freed from accompanying perchloric acid by a second separation on the same column, with water as eluent. After elution with acetone, the SXO was crystallized by evaporation.     

Very high-pressure capillary liquid chromatography assisted by voltage

Capillary liquid chromatography at moderately high pressures and capillary electrochromatography (CEC) have been combined to drive the mobile phase through capillary columns packed with small diameter particles. In a column packed with 1.5 microm nonporous particles, linear velocities near 3mm/s were observed when combining inlet pressures of 690 bar (10,000 psi) and an applied voltage of 25 kV. Optimum linear velocity for the column was achieved using a pressure-voltage combination of 350 bar (5000 psi) and 5 kV. Separation efficiencies at near optimum linear velocity agreed with those predicted by the van Deemter equation for liquid chromatography. Retention factors were observed to decrease under pressure-voltage combination as the voltage was increased; such a behavior has been attributed to Joule heating effects.     

Optimization of two-dimensional gradient liquid chromatography separations

An almost orthogonal comprehensive two-dimensional liquid chromatography was developed for the separation of phenolic and flavone natural antioxidants by using combinations of a polyethylene glycol silica micro-column in the first dimension and a porous-shell fused-core C18 column in the second dimension, both in the reversed-phase mode. System orthogonality was improved using parallel gradients of acetonitrile in buffered mobile phase. A new approach was proposed to optimize matching segmented gradient profiles in the two dimensions. An algorithm was developed for automatic corrections of the shifts in retention in the second dimension induced by the parallel two-dimensional gradient operation technique. Using the porous-shell C18 column in the second dimension at elevated temperature (60 degrees C) and high pressure (480 bar) with optimized segmented profiles of the parallel gradients in the two dimensions, the overall separation time for comprehensive LC x LC was reduced to 30 min.     

Gradient separations of complex mixtures by micro high-performance liquid chromatography

High-performance micro packed fused-silica and open-tubular glass capillary columns were prepared and applied to separations of complex mixtures. Solvent-gradient elution proved quite useful for the separation of solutes with wide polarity. Instruments and some applications are described.     

Determination of steroids by liquid chromatography/mass spectrometry

On-line atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) liquid chromatography/mass spectrometry (LC/MS) were evaluated for the analysis of a variety of steroids. Steroids were classified into three major groups based on the spectra and the sensitivities observed: (I) those containing a 3-one, 4-ene functional group, (II) those containing at least one ketone group without conjugation, and (III) those containing hydroxy group(s) only. In the APCI mode, the best sensitivity and the lowest detection limit for all three groups were obtained by using a mobile phase consisting of methanol and 1%–2% acetic acid in water. The APCI spectra were characterized by MH⁺, MH⁺-H₂O, MH⁺-2H₂O, etc., with the degree of H₂O loss being compound dependent: group I steroids produced stable MH⁺ and group III steroids showed extensive water loss. In the electrospray mode the best sensitivity and the lowest detection limit for the first two groups were obtained when pure methanol and water were used as the mobile phase. This condition produced abundant stable MNa⁺ due to ubiquitous sodium. Detection limits in the 5–15 pg range can be easily achieved using ESI LC/MS. Addition of ammonium acetate or use of acetonitrile in the mobile phase, common in the LC/MS analysis of steroids, decreased the sensitivity for the group I and II steroids and thus should be avoided. For group III steroids, the detection limit can be improved by the addition of acetic acid to the mobile phase.     

High-pressure liquid chromatography of steroids.

After a brief discussion of the merits and limitations of high-pressure liquid chromatography (HPLC) relative to other chromatographic methods, special problems in the application to steroids are discussed. Publications on HPLC of steroids are then discussed under the headings of individual classes, arranged generally in the order of increasing polarity.