a) Regarding HPLC, explain specifically why it is vital that the mobile phase (C
ID: 633562 • Letter: A
Question
a) Regarding HPLC, explain specifically why it is vital that the mobile phase (Carrier) is either held under a slight vacuum or is purged with helium gas before entering the HPLC column.
b) An analysis of fatty acids is desired. These fatty acids have boiling points in excess of 275O C. The sample is extracted with methanol to obtain a solution that only contains the fatty acids. Which of the following Methods/Cases is not possible and why, & which is the best approach & why?
Case I-The methanol extract is mixed with a compound that reacts with the fatty acids and adds a methyl group to the end of the fatty acid
Explanation / Answer
Chromatography, a group ofmethods for separating very small quantities of complex mixtures, with very high resolution, is one of the most important techniques in environmental analysis. The ability of the modern analytical chemist to detect specific compounds at ng/g or lower levels in such complex matrices as natural waters or animal tissues is due in large part to the development of chromatographic methods.
The science of chromatography began early in the twentieth century, with the Russian botanist Mikhail Tswett, who used a column packed with calcium carbonate to separate plant pigments. The method was developed rapidly in the years after World War II, and began to be applied to environmental problems with the invention of the electron capture detector (ECD) in 1960 by James Lovelock. This detector, with its specificity and very high sensitivity toward halogenated organic compounds, was just what was needed to determine traces of pesticides in soils, food and water and halocarbon gases in the atmosphere. This happened at exactly the time when the effect of anthropogenic chemicals on many environmental systems was becoming an issue of public concern. Within a year, it was being applied to pesticide analysis. The pernicious effects of long lived, bioaccumulating pesticides, such as DDT, would have been very difficult to detect without the use of the ECD. The effect of this information on public policy has been far-reaching.
The basis of all types of chromatography is the partition of the sample compounds between a stationary phase and a mobile phase which flows over or through the stationary phase. Different combinations of gaseous or liquid phases give rise to the types of chromatography used in analysis, namely gas chromatography (GC), liquid chromatography (LC), thin layer chromatography (TLC), and supercritical fluid chromatography (SFC).
Chromatography has increased the utility of several types of spectroscopy, by delivering separate components of a complex sample, one at a time, to the spectrometer. This combination of the separating power of chromatography with the identification and quantitation of spectroscopy has been most important in environmental analysis. It has enabled analysts to cope with tremendously complex and extremely dilute samples.
In order to be able to determine very small amounts (very low concentrations) of chemicals in
the environment, it is necessary to follow a series of operations :
1) isolation (extraction and separation) of the target chemicals from sample matrix (air, water,
sediment, living things, etc.).
2) separation and purification of the target chemical from co-extracted, non-target chemicals
(sample clean-up).
3) sample concentration.
4) measurement by highly selective and sensitive analytical equipment, such as gas
chromatography / mass spectrometry (GC/MS).
derivatise (chemically modify) non-volatile and heat degradable target chemicals prior to
analysis by the two most commonly used instrumental methods in environmental analysis,
GC or GC/MS.
The following sections describe this series of analytical operations in more detail, and introduce
some of the main analytical methods for environmental pollutants.
Occasionally it is also necessary to
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