Electronic Supplementary Material
Effect of the extraction solvent type and volume
The selection of an appropriate solvent is of high importance for the DLLME process. It is based on its higher than water density, its extraction capability of the target compounds, its good gas chromatographic behavior and its low solubility in water. Most of the dense solvents are halogenated solvents. According to the strong response with a high peak tailing of the halogenated solvents in ECD, there are some restrictions for choosing a suitable extraction solvent. Among all the solvents, carbon disulfide (CS2) (density: 1.2 g mL−1, boiling point: 46 °C), illustrating all these properties, and chlorobenzene (density: 1.1 g mL−1, boiling point: 131.6 °C), displaying a much lower response factor than the analytes in ECD, were selected as extraction solvents and tested for the DLLME procedure to extract 5-nitropiazselenol from the water samples.
For evaluating the effect of the extraction solvent on the extraction efficiency, 5-nitropiazselenol was formed in the screw cap glasses as already described. The analysis was then conducted with 0.50 mL of ethanol, containing different volumes of the extraction solvent to achieve a 5.0 μL volume of the sedimented phase. In fact, 20.0 μL carbon disulfide and 11.0 μL chlorobenzene were required for this aim, resulting in enrichment factor values of 34 ± 4.1 and 122 ± 3.9 (n = 3), respectively. According to these results, chlorobenzene presented a higher extraction efficiency (this was probably due to the interaction between the benzene ring of chlorobenzene and the 5-nitropiazselenol ring). Therefore, chlorobenzene was selected as the optimum extraction solvent.
To examine the effect of the extraction solvent volume on the 5-nitropiazselenol extraction from the 5.00 mL water samples, solutions containing different chlorobenzene volumes were subjected to the same DLLME procedures. The experimental conditions were fixed and included the use of 0.50 mL ethanol, containing various chlorobenzene volumes from 11.0 to 31.0 μL. As a result, the volume of the sedimented phase increased from 5.0 to 25.5 μL. Fig. S1 displays the curve of the enrichment factor versus the volume of the extraction solvent (chlorobenzene). In line with this Figure, the enrichment factor decreases with increasing the chlorobenzene volume, owing to the increase of the sedimented phase volume. Subsequently, at low volumes of the extraction solvent a high enrichment factor was obtained. Thereby, an improvement in sensitivity was achieved using 11.0 μL of chlorobenzene. In terms of using volumes lower than 11.0 μL, the sedimented phase volume would be less than 5.0 μL, causing difficulties in its removal with a microsyringe and encountering systematic errors.
Fig.S1. Effect of the chlorobenzene (extraction solvent) volume on the enrichment factor of the selenium derivative obtained from DLLME. Derivatization and extraction conditions: water sample volume, 5.00 mL; pH = 2; Se(IV) concentration, 2.00 μg L−1; 4-nitro-o-phenylendiaminein amount, 0.00025 g; derivatization time, 7 min; derivatization temperature, 75 ºC; disperser solvent (ethanol) volume, 0.50 mL.
Influence of the disperser solvent type and volume
The disperser solvent should dissolve the extraction solvent. In addition, it should be miscible in water. Solvents such as acetone, acetonitrile, methanol and ethanol were used to investigate the effect of these solvents on the DLLME performance. A series of the sample solutions were studied with the employment of 0.50 mL of each disperser solvent, containing 11.0 μL of chlorobenzene (extraction solvent). The obtained enrichment factors for acetone, acetonitrile, methanol and ethanol were 118 ± 3.8, 123 ± 3.6, 110 ± 4.1 and 122 ± 3.9 (n = 3), respectively. The results exhibited no considerable differences between the disperser solvents; however, the smaller ethanol toxicity made ethanol a better choice.
The influence of the disperser solvent amount on the extraction efficiency was tested over the range of 0.25-1.50 mL, but the variation of the ethanol volume (disperser solvent) caused changes in the sedimented phase volume. Hence, it is impossible to consider independently the influence of the ethanol volume on the extraction efficiency in DLLME. To avoid this problem and in order to attain a constant volume of the sedimented phase, the ethanol and chlorobenzene volumes were changed simultaneously. The experimental conditions were fixed and included the use of different ethanol volumes: 0.25, 0.50, 0.75, 1.00, 1.25 and 1.50 mL containing 10.5, 11.0, 12.0, 14.0, 15.5 and 17.5 μL of chlorobenzene, respectively. Under these conditions the sedimented phase volume was constant (5.0 ± 0.2 μL). In line with the results (Fig. S2), initially the extraction efficiency increases and, afterwards, it reduces as the ethanol volume is increased. This observation can be attributed to the fact that at lower ethanol volumes, the cloudy suspension of chlorobenzene droplets is not formed well, resulting in a decrease in the extraction recovery. At higher ethanol volumes, the 5-nitropiazselenol solubility in water increases and the extraction efficiency decreases. Hence, the ethanol volume of 0.50 mL was selected as the optimum volume for the disperser solvent.
Fig. S2. Influence of the disperser solvent volume (ethanol) on the enrichment factor of the selenium derivative obtained from DLLME. Derivatization and extraction conditions: water sample volume, 5.00 mL; pH = 2; Se(IV) concentration, 2.00 μg L−1; 4-nitro-o-phenylendiaminein amount, 0.00025 g; derivatization time, 7 min; derivatization temperature, 75 ºC; extraction solvent (chlorobenzene) volume, 11.0 μL.
Effect of the extraction time
The extraction time is one of the salient factors in most of the extraction procedures, especially in microextraction methods such as SPME and LPME. In DLLME, the extraction time is defined as the time that the cloudy solution is formed until the start of centrifugation. The dependence of the extraction efficiency upon the extraction time was studied within the range of 5 sec – 60 min. The results indicated that the extraction time had no significant effect on the extraction efficiency. The peak area is independent of extraction time up to 1 hour. It was revealed that after the formation of the cloudy solution, the surface area between the extraction solvent and the aqueous phase (water sample) was infinitely large. The 5-nitropiazselenol transition from the aqueous phase to the extraction solvent was fast enough to lead to a quick achievement of the equilibrium state. Consequently, the extraction times could be very short. This is one of the most important advantages of the DLLME technique. Thus, 5 sec of extraction time was selected as the optimum of extraction time in order to save time without reducing the extraction efficiency.