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High Performance Liquid Chromatography - Lough W.J.

Lough W.J. High Performance Liquid Chromatography - Blackie academic, 1977. - 282 p.
Download (direct link): highperoranceliquidchromatographi1977.pdf
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8.5 Examples of applications
Some examples of common methods are detailed below along with general notes on the choice of the sample preparation procedure and the factors influencing the choice.
8.5.1 Tamoxifen in plasma
The requirement is for a method capable of measuring this anti-cancer drug down to 20ng/ml. The structure is shown in Figure 8.4a. The drug is lipophilic so solvent extraction is possible. The basic functional group means that high pH is necessary for extraction. Endogenous compounds such as proteins and fats would cause rapid deterioration of HPLC columns if the sample were injected directly. The method involves raising the pH of the plasma (1 cm3) using a small volume of ammonium hydroxide and then extracting with diethyl ether. The ether layer is then
(CH,) 2 CH Ř\^ N 'V"y/C1 N
Figure 8.4 Chemical structures: (a) tamoxifen; (b) butylated hydroxytoluene; (c) atrazine.
evaporated to dryness under nitrogen (very easily done as the solvent is volatile). The extracts are then reconstituted in 0.1ml of mobile phase so that a tenfold concentration factor is achieved.
8.5.2 Butylated hydroxytoluene in animal diet
The requirement is a method capable of measuring this anti-oxidant down to 250 mg/kg. The compound (Figure 8.3b) contains an aromatic group and is suitable for HPLC with UV detection. The solid matrix requires some kind of extraction to release the analyte into a liquid (preferably volatile) phase. The concentrations required are quite high but the matrix
will contain many fatty compounds that could cause the HPLC column to deteriorate rapidly. The method involves taking 10 g of sample, adding 100 cm3 of methanol (in which the analyte is very soluble due to the hydroxy group) and homogenising for 1 min. An aliquot of the supernatant is then taken and evaporated to dryness under nitrogen and redissolved in HPLC mobile phase. This very simple sample preparation procedure is possible only because of the high concentrations involved. If a lower limit of detection were required, the probable approach would be to use a Soxhlet extraction to ensure quantitative recovery of analyte at the very low levels. This would co-extract a lot of other material and so further clean-up using liquid-liquid extraction and SPE would be required.
8.5.3 Pesticides in water
Pesticides such as atrazine (Figure 7.4c) are determined in water down to the EC drinking water limit of 100ng/l. Atrazine is a basic compound so liquid-liquid extraction at high pH is possible. However, solid phase extraction using Cl8 or C8 bonded silica is now preferred. Typically, the cartridge is ‘primed’ by passing through a small volume of methanol, the sample (100 cm3) is loaded and analyte retained due to the hydrophobic nature of the cartridge and the aromatic ring on the analyte. The analyte is then eluted from the cartridge by a small volume (say 1 cm3) of a semi-polar solvent such as ethyl acetate, and injected directly onto the HPLC. A 100-fold concentration factor is then achieved.
8.6 Automation
As stated earlier, the rate-limiting step of many assays is the sample preparation. Not surprisingly, much effort has been devoted to attempts to automate sample preparation where procedures are lengthy and where many samples are analysed. Successful automation can lead to a greater throughput of samples, more accurate methods, cost savings and can free laboratory staff to undertake more interesting and productive work. Automation of instrumentation and data handling is much easier than automation of sample preparation since instruments and data are used for many different types of analysis. Sample preparation procedures depend on both the matrix and the analyte properties and are therefore rather more application specific.
Solid phase extraction systems have been successfully automated (for example in the ASPEC system, marketed by Gilson). This allows samples to be applied to the cartridge and is linked on-line to the HPLC. An extension of this is the ASTED which combines dialysis with trace enrichment on a solid phase and is automatically coupled to the HPLC. These
methods have found widespread application for the analysis of drugs and metabolites in body fluids. The fact that solid-phase extraction methods may potentially be automated is one further reason for their current popularity in the manual mode; if many samples are expected the manual method can be automated. This is much more difficult with liquid-liquid extraction.
Another approach to automation has been the development of robots or more correctly robotic arms. These are reprogrammable mechanical arms which can carry out most of the tasks that humans carry out, but of course can handle toxic substances and can work longer hours in a continuous period. Most robotic configurations consist of the control arm with a series of workstations in a circle around the processing arm. The arm moves from one workstation to the next carrying out its sampling tasks. Adding reagents, solvent extraction, centrifugation, phase separation and transfer to HPLC autosampler have all been proven possible.
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