Development of a thermospray nebulizer interface for liquid chromatography with flame ionization detection and detector response studies of volatile and non-volatile compounds

Abstract

Liquid chromatography (LC) is the analytical tool of choice for the investigation of by-products in pharmaceutical and chemical industry. The UV- and refractive index detector (RFID) encounter to the most common detectors for these purposes. The detection of unknown by-products deliver no sufficient information, since both detectors only can be used for a limited spectrum of analytes. Furthermore, without knowledge of the chemical structure, a semi-quantitative analysis is barely possible. \nThe flame ionization detector (FID) is advantageous to provide semi-quantitative data in the detection and quantification of, e.g., synthesis by-products in pharmaceuticals and chemical products. In contrast to commonly applied detectors, the FID signal is proportional to the carbon content of the analyte and allows the estimation of an analyte concentration directly out of the obtained signal. Several attempts to employ the FID for liquid chromatography have been carried out until yet [1]. \nThis thesis described the development of a novel LC/FID interface and a FID response study of more than 100 compounds in order to achieve a better understanding of differences between gas chromatography (GC)/FID and LC/FID response data. The thesis was divided into 3 major sections: 1. a detailed study of previously described interfaces, 2. the development and optimization of a novel interface and 3. the analysis and comparison of response data obtained by GC/FID and LC/FID. \nThe historical conveyor type systems were designed to overcome the problems arise in FI detection by use of organic solvents in liquid chromatography. Therefore the systems dependent choice of non-volatile analytes in the beginning of LC/FID coupling change to volatile analytes by implementation of only water liquid chromatography [1]. Previously invented interfaces were studied to find out the advantages and disadvantages between the different types of interfaces, such as conveyor based interfaces, capillary jet interfaces or spray chamber interfaces. The focus was set on direct coupling techniques which can be operated without pre-evaporation steps of signal disturbing organic solvents, as known from conveyor type interfaces. The advantages and disadvantages of these interfaces were critically reviewed to design and present a novel interface. \nIn the development and optimization of the here designed interface the influence of the nebulizer material on flame stability and capillary blockage was shown. Previously reported problems such as blocking of the transfer capillary were solved using a stainless steel nebulizer body and transfer capillary. The effects of the working parameters such as backpressure, gas flow, distance between nebulizer nozzle and FID collector or FID temperature on the signal were analyzed. \nThe novel interface was validated for selected compounds known from literature. The linear correlation of the concentration and obtained FID signal of 21 N-heterocycles and 6 alcohols was found to be R² = 0.991 to 0.999. The limits of detection of N-heterocycles ranged from 0.24 ng (pyrimidine-N-oxide) to 1.26 ng (s-triazin) absolute injected carbon. Obtained results for chromatographic separation of the alcohols propanol, butanol, pentanol and hexanol, used within previous studies in the field of LC/FID coupling were presented and compared to literature. The developed interface showed a substantial improvement of the absolute injected carbon concentration down to 0.28 ng (ethanol) in comparison to previously invented interfaces. \nThe use of different theoretical and practical response models was discussed to assist the response studies performed in the final section of the thesis. The scope was set on the experimental derived Effective Carbon Number (ECNExp) [2, 3] and experimental relative sensitivity (RFExp) [2, 4], respectively. \nThe influences of functional groups and substitutes, such as hydroxyl groups, halogens and ketones were discussed regarding former GC/FID studies. For some functional groups, effects on the obtained signals were found to be more negligible by extension of the carbon backbone. The influence of mono-alcohols decreased from C1 to C7. The effects of mono-, di- and poly-substituted compounds were compared and a substantial effect of the substitute location and the response factor was observed for, e.g., butan-1,2-ole. \nInflame processes for pyrimidine and pyridazine were proposed by the support of literature data. The response data of structural isomers obtained by theoretical response models (e.g. pyridine) were compared to experimental data of literature and within this study. The differences of theoretical isomer response data (e.g. pyridine, pyrimidine) to experimental data were explained by the occurring inflame processes. In the end the response data were compared to GC/FID response data as far as they are available. \nThe present work is a great step forward for the analysis of volatile and non-volatile compounds using LC/FID. For the first time the responses of more the 100 different compounds were detected using LC/FID and compared to GC/FID response data if available. Much more LC feasible compounds are available, therefore further work is necessary to investigate all of this compounds and compare them to GC/FID responses. Further research in pharmaceutical industry is required to show the advantages of the semi-quantitative analysis of by-products by LC/FID. \nReferences \n[1] C. Becker, M.A. Jochmann, T.C. Schmidt, An Overview of Approaches in Liquid Chromatography Flame ionization Detection, TrAC Trends in Analytical Chemistry, 110 (2018) 143-149. \n[2] J.T. Scanlon, D.E. Willis, Calculation of Flame Ionization Detector Relative Response Factors Using the Effective Carbon Number Concept, J. Chromatogr. Sci., 23 (1985) 333-340. \n[3] J.C. Sternberg, Gallaway, W.S. and Jones, D.T.L., The Mechanism of Response of Flame Ionization Detectors, in: Gas Chromatography: Third International Symposium Held Under the Auspices of the Analysis Instrumentation Division of the Instrument Society of America, June 13-16, 1961, Academic, 1962, pp. 231-267. \n[4] W.A. Dietz, Response Factors for Gas Chromatographic Analyses, J. Chromatogr. Sci., 5 (1967) 68-71.