Choice of Column

For more than a decade, since the first application of GC for bile acid analysis by Vanden Heuvel et al. in

1960, GC was performed using metal or glass-packed columns. Introduction of capillary or open tubular columns was a major landmark in the development of GC. The separation of serum bile acids using capillary columns was first demonstrated by Laatikainen et al. in 1975 (Table 1). Coupled with the fact that the retention times are highly reproducible, capillary columns have fast become columns of choice for most chromatographic applications for bile acids. The usual capillary column is 25-50 m long with an internal diameter of 0.22-0.25 mm. The inner wall is coated with the liquid phase, which may vary from nonpolar (e.g. methyl silicones, such as OV-1, CP-Sil-5 CB and SE-30) to more polar (like phenyl-methyl- and cyanopropylsilicone, e.g. CP-Sil-19 CB) depending on the need. Usually peaks are very sharp; however, the dead volume must be very small and

Table 1 Important advances in gas chromatography of bile acids

1960-61 Vanden Heuvel etal.

1965

1975 1987 1998

Grundy et al.

Laatikainen etal Child etal. Batta et al

First application of gas chromatography for bile acid analysis. Bile acid methyl ester-

trifluoroacetates were analysed on packed columns Comprehensive method for faecal bile acid analysis using packed columns. Bile acids were isolated free from faecal fatty acids, sterols and other contaminants before chromatography. Method often used as a standard to which other methods are compared Introduction of capillary columns for serum bile acid analysis Simultaneous quantitation of faecal bile acids, sterols and fatty acids Bile acid analysis was greatly simplified. Bile acidswere derivatizedand quantitated directly in the stool sample and the multiple steps of removal of sterols and fatty acidswere avoided only microgram or nanogram quantities of compounds can be injected in order to get appropriate peak shapes.

New instrumentation and use of splitless injectors have greatly reduced the dead volume while increasing the sensitivity, so that capillary columns are ideal for chromatography of trace compounds in biological fluids. Quantitation of bile acids in a sample is carried out by comparison of peak areas with those of known amounts of reference compounds in a range where a linear detector response is obtained. Usually a known amount of an internal and/or external standard is added to the sample to facilitate quantitation. Often a mass spectrometer is used as a detector for GC, and gas chromatography-mass spectrometry (GC-MS) has the added advantage that the mass fragmentation can give insight to the structure of the compound. The sensitivity of GC-MS is several-fold increased by using selected-ion monitoring, when picomole quantities of bile acids can be detected. Selection of the right internal standard is important for quantitation of bile acids using the mass spectrometer in the selected-ion mode. Often isotope-labelled bile acids (polydeuterated) are used, the advantage being that such compounds mix with the bile acid to be quantitated and are similarly extracted from the biological source. Otherwise, a bile acid with different GC retention time is employed, and peak heights are calibrated using known amounts of the compounds to be quantitated. The mass ion selected for quantitation is usually a high mass ion and with significant abundance.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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