An overview of the chemistry, manufacture, environmental fate and detection of carbofuran - Carbofuran and Wildlife Poisoning: Global Perspectives and Forensic Approaches - page 15

based on the use of a very expensive piece of equipment (upwards of 100 000 USD) and on having a
highly trained operator who can maintain and run it. In some regards, TLC could be viewed as being
more sensitive than other confi rmatory means of analyses. Samples of unknowns are often initially
analysed by TLC because, even in the absence of the parent compound, the presence of metabolites
and breakdown products could be detected, which would not necessarily be the case with GC/MS.
However, TLC will only establish the presence or absence of a carbamate or organophosphorus
compound, hence, the sample must then be analysed using more specifi c confi rmatory methods such
as GC/MS or LC/MS.
TLC consists of applying a small spot of sample approximately 1.5 cm from the bottom of a plate.
The position of the base of the spot is marked with a pencil. The plate will either be a sheet of glass,
metal or rigid plastic coated with a thin layer (hence the name) of absorbent material which tends to
be silica or alumina-based. When a fl uorescent compound is present within the thin layer material,
the plate fl uoresces everywhere except where there is an organic compound.
Ideally a mixture of known compounds (containing the compound or class of compounds sus-
pected to be in the sample) is added to the same plate for comparison.
Once treated with the unknown sample and the standard control mixture the plate is dried so
that all the sample solvent is evaporated. Solvent is then added to a developing chamber in which the
plate will be housed. A piece of fi lter paper is generally placed in the solvent, against the wall of the
chamber, which is then sealed for a certain period of time to ensure saturation of the chamber with
the solvent. This is to prevent the solvent evaporating from the plate as it moves up the column. Once
the plate is placed within the chamber, the solvent is allowed to touch the bottom of the plate, but
cannot go above the pencil line (i.e., be in direct contact with the sample spot). The solvent serves as
the mobile phase, fi rst dissolving the compound(s) in the sample spot and then being drawn up the
plate by capillary action. The material coating the plate serves as the stationary phase.
Separation of the compounds within a sample is achieved due to differences in both their solubil-
ity and their affi nity or absorption to the plate itself. The retention factor (denoted as the R f value)
measures how quickly the compound progresses up the plate relative to the front, i.e., the solvent or
mobile phase. If compounds are coloured, then the components are readily discernible to the naked
eye. If not, the plate is viewed in darkness with a UV lamp. The compounds then show up as dark
spots on the plate which are circled in pencil. On occasion, the sample will run as a streak or a smear
rather than create distinctive spots, making interpretation diffi cult. If no spots are seen on the plate,
then the sample may not be concentrated enough.
TLC is especially useful if a large number of samples must be analysed but few resources are
available, i.e., to pinpoint which samples warrant further analysis. In principle, the technique can be
used to identify the presence of carbofuran metabolites. Ultimately though, a positive screen result
by TLC must still be followed up by confi rmatory methods such as GC/MS or HPLC-MS so that the
presence of the compound of interest can be specifi cally and conclusively confi rmed.
1.5.1.7 ELISA-based method
Immunoassays such as enzyme linked immunosorbent assays (or ELISAs) use antibodies which
react specifi cally to the analyte in a sample rather than binding or adsorbing to it (Harris 2007).
As such, ELISAs could be used to pre-screen samples for presence/absence of carbofuran. First,
the analyte contained in the sample or used for calibration is incubated with a polymer-bound
antibody, generally on a 96 well microtiter plate (Nelson and Cox 2008), to form a complex which
binds to the plate. The fraction of immobilised antibody that binds to the analyte is proportional
to the concentration of the analyte in the unknown sample (Harris 2007). The surface of the plate
is then incubated with a solution of nonspecifi c protein, generally casein or bovine serum albu-
min. This blocks any sites on the plate that do not already have anything bound to them (Nelson
and Cox 2008). The complex formed by the antibody and the analyte (of high molecular weight,