06-11-2012, 06:05 PM
Comparison of Two Detection Methods in Thin Layer Chromatographic Analysis of Some Herbicides in a Coastal Savanna Soil in Ghana
[attachment=38356]
Abstract
o-tolidine plus potassium iodide and photosynthesis inhibition detection methods were investigated for the analysis of three triazine
herbicides (atrazine, ametryne, simazine) and two urea herbicides (diuron, metobromuron) in a coastal savanna soil using thin layer
chromatography to compare the suitability of the two methods for the study of the herbicides. This was done by spiking 5 g of the soil sample
with specific amount of the herbicide standards to generate herbicide-soil concentration of 40.24, 41.46, 40.28, 39.90 and 40.64 μg/g for
atrazine, ametryne, simazine, diuron and metobromuron, respectively. Extraction was performed with acetone/hexane mixture (4:1) and the
detection limit of each herbicide was then determined. In all, the photosynthesis inhibition method performed better for both the triazine and
the urea herbicides, while the o-tolidine plus potassium iodide method was suitable for only the triazine herbicides. With the photosynthesis
inhibition method, detectability in the range of 0.004–0.008 ± 0.002 μg/g was attained for the herbicides using the unclean extracts. In the
case of o-tolidine plus potassium iodide method, detectability of 0.008–0.406 ± 0.02 μg/g was obtained. With the clean up extracts
detectability in the range of 0.025–0.162 ± 0.004 μg/g was obtained using the photosynthesis inhibition method. However, metobromuron
was not detected in the cleaned up extracts when o-tolidine plus potassium iodide detection method was used. For the methods described,
clean up with SPE cartridge, equipped with C-18, is not critical to obtain the desired results.
Introduction
Herbicides belong to the class of pesticides that are used to control weeds. They are, therefore, also called weed
killers. After application of herbicide on target weeds, the active ingradient is gradually lost as a result of
breakdown, evaporation and leaching, and the herbicide residue is the amount that remains on the field after
application and usage (Afful, 2002). While some herbicides have long residual activity and, therefore, persist in the
environment for long time, others have low residual activity and disappear from the environment, or produce low
residual concentrations (Walker, 1973).
The residue level of the herbicide in the soil after application is an important factor to be taken into account when
assessing their performance as weed killers. There is the need to have information about the duration of
phytotoxicity in order to avoid the possibility of damaging a succeeding crop, which may not be tolerant to the
herbicide. The environmentalist needs similar informa-tion to assess the impact of the residue on non-target
organisms either directly or as a consequence of vegetation changes affecting the ecosystem (Helling et al., 1978).
Against this background, it is necessary to study the residue level and the fate of these agro-chemicals after
application on the field to have a better understanding of their behaviour on the environment.
Materials and methods
Chemicals and reagents
The herbicide standards, which were of 98–99.5% purity, were obtained from Dr Ehrenstorfeer, Gmbh. The other
chemicals used in the study were obtained from Merck, Germany and Fluka, Switzerland. They were of analytical
grade and were used without further purification.
The TLC detection reagents for the herbicides were prepared as follows:
o-tolidine + potassium iodide (oTKI) reagent was prepared by dissolving 0.5 g of o-tolidine in 100 μl of glacial
acetic acid and thoroughly mixed with 10 ml of 20% KI solution. The resultant solution was diluted to 500 ml with
distilled water. Photosynthesis inhibition reagent was prepared by mashing 30 g of Panicum maximum and 5 g of
sea sand in a mortar with pestle, 15 ml of distilled water and 3 ml of glycerine were added, mixed thoroughly, and
the liquid squeezed through a knapsack into 50 ml flask. 20 ml of this was added to 13 ml of DCPIP reagent, which
was prepared by dissolving 0.1 g of 2, 6-dichlorophenol-indolphenol sodium salt in 250 ml of borax solution, which
was also prepared by dissolving 3.325 g of borax in 175 ml of distilled water, and the solution was added to 75 ml
of 0.1 M HCl.
Determination of limit of detection
Spiking of soil samples. 5 g of the soil in five extraction flasks, labeled S1, S2, S3, S4 and S5, were spiked with 2 ml
of standard herbicide solution of atrazine, ametryne, simazine, diuron and metobromuron, respectively, to generate
spiking levels of 40.24, 41.46, 40.28, 39.90 and 40.64 μg/g. The concentrations of the herbicide solutions used for
the spiking were 100.59, 103.66, 100.70, 99.75 and 101.59 μg/ml for atrazine, ametryne, simazine, diuron and
metobro-muron, respectively. The spiked soil in the extraction flasks was allowed to stand for 30 min before
extraction was performed.
Extraction. Extraction was performed by adding 20 ml of acetone/hexane mixture (4:1) unto each of the spiked
soil samples and mechanically mixed on a flask shaker for 2 h. Filtration was carried out by use of Whatman No. 42
filter paper. After filtration, the residue was washed three times with 3 ml of the solvent, and the washings were
added to the filtrate, which was then dried over anhydrous sodium sulphate (Na2SO4). The filtrate was concentrated
to dryness by gently blowing in streams of air from a hand dryer. The recovered unclean herbicides were
redissolved in 5 ml of acetone and subjected to thin layer chromatographic analysis.
Clean-up of extracts. The extraction procedure was repeated, but this time the unclean filtrates were cleaned by
passing the filtrates through SPE cartridge equipped with C-18 as adsorbent, which was earlier preconditioned with
2 μl of acetone/water (1: 9). The cartridge and its contents were dried for 15 min by a vacuum pump. The herbicide
was then eluted with 10 μl of acetone to give the clean extracts. The clean extracts were concentrated to 5 μl using
the hand dryer and then subjected to thin layer chromatographic analysis.
Results and discussion
TLC analysis of extracts
The Rf values, which is an identification parameter obtained for the herbicides, are presented in Table 1. The results
suggest that in a multi-residual procedure involving a mixture of these chemicals, the silica gel-ethyl acetate system
used for the investigation could not be very useful for analysis of atrazine, ametryne and metobromuron, as their
spots would overlap and resolution would be difficult. This is because they have Rf values that are close (Table 1),
particularly atrazine and ametryne. However, for samples known to contain one of these chemicals, the system
could conveniently be used. The Rf values obtained compare favourably with the findings of (Lowor et al., 2000).
They reported Rf values of 0.61, 0.61, 0.57 and 0.41 at 32 0C for atrazine, ametryne, metobromuron and diuron,
respectively.
Conclusion
The results suggest that the photosynthesis inhibition method is a better detection method for both the triazine and
the urea herbicides, whilst the o-tolidine plus potassium iodide method is suitable for only the triazine herbicides
used in the investigation. For the methodology described, clean up with SPE cartridge equipped with C-18 as
adsorbent is not critical to obtain the desired result.
[attachment=38356]
Abstract
o-tolidine plus potassium iodide and photosynthesis inhibition detection methods were investigated for the analysis of three triazine
herbicides (atrazine, ametryne, simazine) and two urea herbicides (diuron, metobromuron) in a coastal savanna soil using thin layer
chromatography to compare the suitability of the two methods for the study of the herbicides. This was done by spiking 5 g of the soil sample
with specific amount of the herbicide standards to generate herbicide-soil concentration of 40.24, 41.46, 40.28, 39.90 and 40.64 μg/g for
atrazine, ametryne, simazine, diuron and metobromuron, respectively. Extraction was performed with acetone/hexane mixture (4:1) and the
detection limit of each herbicide was then determined. In all, the photosynthesis inhibition method performed better for both the triazine and
the urea herbicides, while the o-tolidine plus potassium iodide method was suitable for only the triazine herbicides. With the photosynthesis
inhibition method, detectability in the range of 0.004–0.008 ± 0.002 μg/g was attained for the herbicides using the unclean extracts. In the
case of o-tolidine plus potassium iodide method, detectability of 0.008–0.406 ± 0.02 μg/g was obtained. With the clean up extracts
detectability in the range of 0.025–0.162 ± 0.004 μg/g was obtained using the photosynthesis inhibition method. However, metobromuron
was not detected in the cleaned up extracts when o-tolidine plus potassium iodide detection method was used. For the methods described,
clean up with SPE cartridge, equipped with C-18, is not critical to obtain the desired results.
Introduction
Herbicides belong to the class of pesticides that are used to control weeds. They are, therefore, also called weed
killers. After application of herbicide on target weeds, the active ingradient is gradually lost as a result of
breakdown, evaporation and leaching, and the herbicide residue is the amount that remains on the field after
application and usage (Afful, 2002). While some herbicides have long residual activity and, therefore, persist in the
environment for long time, others have low residual activity and disappear from the environment, or produce low
residual concentrations (Walker, 1973).
The residue level of the herbicide in the soil after application is an important factor to be taken into account when
assessing their performance as weed killers. There is the need to have information about the duration of
phytotoxicity in order to avoid the possibility of damaging a succeeding crop, which may not be tolerant to the
herbicide. The environmentalist needs similar informa-tion to assess the impact of the residue on non-target
organisms either directly or as a consequence of vegetation changes affecting the ecosystem (Helling et al., 1978).
Against this background, it is necessary to study the residue level and the fate of these agro-chemicals after
application on the field to have a better understanding of their behaviour on the environment.
Materials and methods
Chemicals and reagents
The herbicide standards, which were of 98–99.5% purity, were obtained from Dr Ehrenstorfeer, Gmbh. The other
chemicals used in the study were obtained from Merck, Germany and Fluka, Switzerland. They were of analytical
grade and were used without further purification.
The TLC detection reagents for the herbicides were prepared as follows:
o-tolidine + potassium iodide (oTKI) reagent was prepared by dissolving 0.5 g of o-tolidine in 100 μl of glacial
acetic acid and thoroughly mixed with 10 ml of 20% KI solution. The resultant solution was diluted to 500 ml with
distilled water. Photosynthesis inhibition reagent was prepared by mashing 30 g of Panicum maximum and 5 g of
sea sand in a mortar with pestle, 15 ml of distilled water and 3 ml of glycerine were added, mixed thoroughly, and
the liquid squeezed through a knapsack into 50 ml flask. 20 ml of this was added to 13 ml of DCPIP reagent, which
was prepared by dissolving 0.1 g of 2, 6-dichlorophenol-indolphenol sodium salt in 250 ml of borax solution, which
was also prepared by dissolving 3.325 g of borax in 175 ml of distilled water, and the solution was added to 75 ml
of 0.1 M HCl.
Determination of limit of detection
Spiking of soil samples. 5 g of the soil in five extraction flasks, labeled S1, S2, S3, S4 and S5, were spiked with 2 ml
of standard herbicide solution of atrazine, ametryne, simazine, diuron and metobromuron, respectively, to generate
spiking levels of 40.24, 41.46, 40.28, 39.90 and 40.64 μg/g. The concentrations of the herbicide solutions used for
the spiking were 100.59, 103.66, 100.70, 99.75 and 101.59 μg/ml for atrazine, ametryne, simazine, diuron and
metobro-muron, respectively. The spiked soil in the extraction flasks was allowed to stand for 30 min before
extraction was performed.
Extraction. Extraction was performed by adding 20 ml of acetone/hexane mixture (4:1) unto each of the spiked
soil samples and mechanically mixed on a flask shaker for 2 h. Filtration was carried out by use of Whatman No. 42
filter paper. After filtration, the residue was washed three times with 3 ml of the solvent, and the washings were
added to the filtrate, which was then dried over anhydrous sodium sulphate (Na2SO4). The filtrate was concentrated
to dryness by gently blowing in streams of air from a hand dryer. The recovered unclean herbicides were
redissolved in 5 ml of acetone and subjected to thin layer chromatographic analysis.
Clean-up of extracts. The extraction procedure was repeated, but this time the unclean filtrates were cleaned by
passing the filtrates through SPE cartridge equipped with C-18 as adsorbent, which was earlier preconditioned with
2 μl of acetone/water (1: 9). The cartridge and its contents were dried for 15 min by a vacuum pump. The herbicide
was then eluted with 10 μl of acetone to give the clean extracts. The clean extracts were concentrated to 5 μl using
the hand dryer and then subjected to thin layer chromatographic analysis.
Results and discussion
TLC analysis of extracts
The Rf values, which is an identification parameter obtained for the herbicides, are presented in Table 1. The results
suggest that in a multi-residual procedure involving a mixture of these chemicals, the silica gel-ethyl acetate system
used for the investigation could not be very useful for analysis of atrazine, ametryne and metobromuron, as their
spots would overlap and resolution would be difficult. This is because they have Rf values that are close (Table 1),
particularly atrazine and ametryne. However, for samples known to contain one of these chemicals, the system
could conveniently be used. The Rf values obtained compare favourably with the findings of (Lowor et al., 2000).
They reported Rf values of 0.61, 0.61, 0.57 and 0.41 at 32 0C for atrazine, ametryne, metobromuron and diuron,
respectively.
Conclusion
The results suggest that the photosynthesis inhibition method is a better detection method for both the triazine and
the urea herbicides, whilst the o-tolidine plus potassium iodide method is suitable for only the triazine herbicides
used in the investigation. For the methodology described, clean up with SPE cartridge equipped with C-18 as
adsorbent is not critical to obtain the desired result.