Gerald Manius

High-resolution reversed-phase liquid chromatography utilizing microbore column concatenation

Abstract It has been demonstrated previously that extremely high efficiencies can be obtained by coupling microbore columns together in series and that efficiency increases linearly with each column addition. Only recently has it become possible to prepare 50 cm × 1 mm I.D. columns packed with 8-μm reversed-phase material exhibiting an optimum plate height of about two particle diameters. A chromatographic system based on the concatenation of these columns, which allows the achievement of a high number of theoretical plates in several hours and which can be arranged with little instrument modification is reported. Experimental data presented in this paper indicate that coupling conventional larger diameter columns results in about 60% loss of column efficiency for each coupling step, whereas coupling microbore columns actually produces efficiencies of 100% for each concatenation. Thus, the design of a very high-resolution, high-efficiency high-performance liquid chromatography system makes the use of microbore columns imperative. This paper deals with both theoretical and experimental aspects of high-resolution liquid chromatography, demonstrates the feasibility of setting up the system, and shows examples of how difficult separations such as for deuterium-labeled isotopic compounds can be achieved easily.

Size exclusion chromatography with Corona charged aerosol detector for the analysis of polyethylene glycol polymer

A technique of using size exclusion chromatography (SEC) with the Corona charged aerosol detector (CAD) was developed and evaluated in comparison with refractive index (RI) and evaporative light scattering detection (ELSD) for fast screening of polyethylene glycol (PEG), a polymer used in preparing pegylated pharmaceutical compounds. These detection techniques were used in the analysis of multiple lots of PEG reagents. CAD was found to provide more accurate impurity and polydispersity profiles of PEG reagents that better differentiate their quality, while RI was not suitable for this application due to its low sensitivity and ELSD led to underestimation of the impurity and polydispersity. The accuracy of polydispersity determination by SEC-CAD was validated against a commercial reference standard of known polydispersity. The SEC-CAD technique and the observed differences between the three detectors can also be applied to polymer analysis in general.

Three Approaches to the Analysis of Trace Formaldehyde in Bulk and Dosage Form Pharmaceuticals

Trace-level determinations for the presence of formaldehyde in both bulk and dosage form pharmaceuticals were developed using three innovative strategies. One system adapted the chromotropic acid spot test for formaldehyde. This was accomplished spectrophotometrically over a linear detection range against authentic control samples. The other two chromatographic approaches necessitated rapid derivatization. One derivative was its corresponding oxime, formaldoxime, which was resolved on a gas chromatographic porous polymer column and sensed by a nitrogen-specific detector. The other derivative, sodium formate, was detected and quantified on an ion chromatograph using an anion-exchange column and a conductivity detector. The chromotropic acid technique was sensitive but not specific for formaldehyde. The chromatographic techniques required a high degree of water solubility. All were subject to interferences that could preclude their use for a particular application. None of the tested samples, which included a penicillin analogue, a pharmaceutical dosage form additive, a vitamin, and biological proteins, showed the presence of formaldehyde at trace levels.

Recycling liquid chromatography using microbore columns

Abstract A study of recycling in high-performance liquid chromatography was carried out using 50 cm × 1 mm I.D. microbore columns. It is demonstrated that, although there are stricter requirements for the recycling microbore column system design than for larger diameter columns, the system can be designed to be highly efficient and can be successfully employed for analytical work. Theoretical aspects of an alternate pumping technique are considered. Equations relating resolution between two peaks of interest and limiting cycle number to various chromatographic parameters are derived. The experimental design of a computerized system operating in an automated mode is described. Several applications demonstrating how closely eluting solutes can be separated are shown and advantages and disadvantages of recycling using scaled down chromatographic systems are discussed.

The quantitation of sugars from differential culture media by gas—liquid chromatography

Certain sugar alcohols and reducing sugars are added to agar or carrageenan culture media for purposes of identifying specific gram-negative bacteria (Enterobacteriaceae). These sugars can be separated and quantitated reliably by the use of gas—liquid chromatography. The reducing sugars, after extraction, are converted to their aldononitrile acetates, whereas the extracted sugar alcohols are converted to their corresponding alditol acetates. Oxalic acid is added in the extraction of the reducing sugars to inhibit side reactions and to prevent chelation. Xylitol is observed to be an effective internal standard. Linearity of response is confirmed by data representing three different levels of sugar content in the culture media.

Identification and determination of thyroxine using gas-liquid chromatography.

Abstract A quantitative gas-liquid chromatographic assay method has been developed for the thyroid hormone thyroxine as a bulk substance. The trimethylsilylated derivative is chromatographed with an internal standard on an OV-101 column by means of flame ionization detection. Analogs of thyroxine can be identified and quantitated as possible contaminants. Electron-capture detection of thyroxine at the picogram level has been demonstrated. Complete silylation has been confirmed.

Sample Preparation Method Validation

The analytical procedure describes all the steps and details associated with performing an analysis. The validation process establishes the performance characteristics of the analytical procedure to meet the output requirements for the intended analytical application. During most validation processes, little attention is spent on sample preparation conditions and their effect on the overall analytical method. Potential approaches and practices commonly used for the sample preparation component of the analytical procedure during the validation process are discussed.