Development of High Resolution Pyrolysis-Gas Chromatography for the Identification of Microorganisms

Abstract

The application of pyrolysis-gas chromatography (PGC) to the rapid identification of medically and economically important microorganisms has increased in the last five years. However, there are major problems concerning the resolution of such complex pyrolyzates and the long term reproducibility of the PGC system. Use of the PGC system described by Shulman et al. (J. Chromatog. Sci. 7, 36, 1969) and optimization of the chromatographic parameters resolved approximately 200 peaks from the pyrolyzates of representative bacteria, yeast, fungus and mycoplasma. Lyophilized samples of 80 µg were accurately weighed in a pyrex pyrolysis tube which was then inserted into the pyrolysis chamber. The samples were heated to 600°C in eight seconds. The entire pyrolysis chamber was disposed of each time, thus eliminating background in subsequent analyses. The pyrolyzate was separated on a 500 ft by 0.03 in. i.d. capillary column coated with 4% Carbowax 20M. Two-stage temperature programming with a helium flow rate of 5.2 cc/min greatly enhanced the resolution. The sensitivity was such that peaks equivalent to 1 ng were readily detected. The reproducibility was excellent initially, but the column exhibited signs of aging after 100 analyses in each direction. Our observations suggested that control of column aging in this system may be aided by use of short disposable precolumns.