# Mussel Inspired Polydopamine as Silica Fibers Coating for Solid-Phase Microextraction
## 3 Results
### Optimization of the SPME Procedure  
Biphenyl and benzo(a)pyrene were used as model compounds to test the performances of the new fiber. Initially, PDA fibers of different thickness were tested conducting extractions on working solution (1 ug/mL) of the two analytes for 30 min at room temperature and under constant stirring (800 rpm). The fibers were then transferred into the SPME/LC interface to be desorbed in dynamic mode by switching the valve from the INJECT to LOAD position after 10 min. As inferable by Figure 4, the extraction efficiencies of the coatings were found to increase with the dopamine concentration used for their preparation, and the thickest PDA fiber was then selected for subsequent experiments. Then, the SPME-LC-UV-DAD analysis of standard solutions (1.0 ug/mL) of the analytes was performed with the selected PDA fiber in different conditions to optimize the extraction parameters, namely sample volume, temperature, ionic strength, and extraction time, before validating the analytical method. The relevant results are reported in Figure 5.  
Figure 4: Extraction efficiencies of PDA fibers of different thickness. Figure 3: Stability test of bare silica fiber tip and PDA:coated silica fiber tip after the three different concentrations of dopamine monomer, tested using reference washing solutions at three different times ((**left**), 0.75 mg/mL, (**center**) 1.5 mg/mL, (**right**) 3 mg/mL). Extraction performed using different volumes (ranging from 1.5 and 15 mL) of standard solutions showed a direct proportionality between sample volume and extraction efficiency (see Figure 5A), clearly indicating 15 mL as the optimal volume. Testing the temperature conditions, as showed in Figure 5B, two different extraction temperatures (20 and 50 \({}^{\circ}\)C) were explored in this case, and the best results were observed at 20 \({}^{\circ}\)C, which was selected as the working temperature for the following experiments. Regarding the salinity of the used buffer, salt addition has often shown variable effects on SPME, usually increasing the extraction efficiency due to the salting-out phenomenon, sometimes showing the opposite behavior. In this case, the presence of sodium chloride (0%, 5%, 10%, 15%, and 30% (\(w/v\))) showed variable effects on the extraction of the two analytes (Figure 5C), leading to a general decrease at 30%, an always negative effect for biphenyl, and positive effects for benzo(a)pyrene between 5% and 15%. The addition of 10% NaCl was then individuated as a good compromise for an efficient simultaneous extraction of the analytes under study. Extraction time profiles were then established by plotting the area counts versus the extraction time, and the relevant extraction kinetics are reported in Figure 5D. Biphenyl kinetics concretely followed huge time-dependent slope variations with respect to benzo(a)pyrene moiety. However, equilibrium conditions were reached after 60 min for both the analytes and since SPME quantitation is feasible even before adsorption equilibrium is reached, an extraction time of 30 min was eventually chosen for further experiments to obtain a considerable gain on the analysis time. Two desorption steps are distinguishable using the on-line SPME/LC commercial interface: static, in which the fiber is soaked in mobile phase in the interface for a certain time before switching the valve in the inject position, and dynamic, when the fiber coating is exposed to the moving stream of mobile phase. These phases must be carefully blended to balance chromatographic efficiency and satisfactory desorption yields. The best conditions were obtained by combining 20 min of static and 6 s of dynamic desorption. To minimize  
Figure 5: Extraction efficiencies of PDA fibers obtained with varying sample volume (**A**), temperature (**B**), ionic strength (**C**), and extraction time (**D**). the carry-over effects, the fibers were soaked in ethanol for 5 min and rinsed with water after each extraction.