Insight into Quantitation of Solid-phase Microextraction
Alam, Md Nazmul
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Solid-phase microextraction (SPME) is a well-known sampling and sample preparation technique used for a wide variety of analytical applications. As there are various complex processes taking place at the time of extraction that influence the parameters of optimum extraction, a mathematical model and computational simulation describing the SPME process is required for experimentalists to understand and implement the technique without performing multiple costly and time-consuming experiments in the laboratory. In this thesis, a mechanistic mathematical model for the processes occurring in SPME extraction of analyte(s) from an aqueous sample medium is presented. The proposed mechanistic model was validated with experimental data. Several key factors that affect the extraction kinetics, such as sample agitation, fiber coating thickness, and presence of a binding matrix component, are discussed. More interestingly, for the first time, shorter or longer equilibrium times in the presence of a binding matrix component were explained with the help of an asymptotic analysis. Parameters that contribute to the variation of the equilibrium times are discussed, with the assumption that one binding matrix component is present in a static sample. Numerical simulation results show that the proposed model captures the phenomena occurring in SPME, leading to a clearer understanding of this process. Therefore, the currently presented model can be used to identify optimum experimental parameters without the need to perform a large number of experiments in the laboratory. A calibration approach based on standard chemicals loaded onto an extraction phase (calibrant-loaded extraction phase, CL-EP) has gained popularity in various areas of sample analysis, such as environmental, toxicological, and tissue sampling research areas. In this thesis, the kinetics of calibrant release and analyte uptake between the sample and extraction phase with a finite-element analysis (FEA) using COMSOL Multiphysics® software package. Effect of finite and infinite sample volume conditions, as well as various sample environment parameters such as fluid flow velocity, temperature, and presence of a binding matrix component were investigated in detail with the model in relation to the performance of the calibration. The simulation results demonstrate the suitability of the CL-EP method for analysis of samples at various sample environments. The calibrant-loaded approach can provide both total and free concentrations from a single experiment based on whether the Kes value being used is measured in a matrix-matched sample or in a matrix-free sample, respectively. Total concentrations can also be obtained by utilizing CL-EP in combination with external matrix-matched calibration, which can be employed to automate the sampling process and provide corrections for variations in sample preparation, matrix effects, and detection processes. This approach is also suitable for very small volumes of sample, where addition of an internal standard in the sample is either troublesome or can change the sample characteristics. Although the outcome of this study is applicable to any sampler based on calibrant-loaded liquid or solid extraction phase method, experimental data using a solid-phase microextraction (SPME) sampler was used to fit our simulation results. The numerical results are in very good agreement with the experimental data reported previously. Moreover, the computational model and numerical simulation presented will aid in the optimization of sampler design and sampling parameters prior to laboratory experiments, which will translate into savings in terms of time and expensive chemicals. Despite the prevalence of porous-particle based coatings used for microextraction techniques, there is inadequate understanding of how extraction parameters influence the extracted amount and quantification of analytes. This is particularly important when extraction is performed with these solid coatings under pre-equilibrium conditions, for instance, with diffusion based rapid calibration approach which is a popular technique for on-site chemical analysis for not requiring any calibration method or internal standards. This study presents a computational model for porous particle-based coatings used in solid phase microextraction. Although the model describes extraction behavior of analytes for both kinetic and equilibrium regime of extraction profile, the critical parameters for the diffusion based rapid sampling were studied using the developed model. Simulations are conducted under variations in both mass transfer and adsorptive surface binding constants, coating capacity, constrained by real-world experimental conditions of finite and infinite sample volume. The model simulation results demonstrated excellent correlation with previously reported experimental data and superior to previous semi-empirical models. In the last chapter of the thesis, a novel SPME coating functionalized with a DNA aptamer for selective enrichment of a low abundance protein from diluted human plasma is described. This approach is based on the covalent immobilization of an aptamer ligand on electrospun microfibers made with the hydrophilic polymer poly(acrylonitrile-co-maleic acid) (PANCMA) on stainless steel rods. A plasma protein, human alpha-thrombin, was employed as a model protein for selective extraction by the developed Apt-SPME probe, and the detection was carried out with liquid chromatography/ tandem mass spectrometry (LC–MS/MS). The SPME probe exhibited highly selective capture, good binding capacity, high stability and good repeatability for the extraction of thrombin. The protein selective probe was employed for direct extraction of thrombin from 20-fold diluted human plasma samples without any other purification. The Apt-SPME method coupled with LC–MS/MS provided a good linear dynamic range of 0.5–50 nM in diluted human plasma with a good correlation coefficient (R2 = 0.9923), and the detection limit of the proposed method was found to be 0.30 nM. Finally, the Apt-SPME coupled with LC–MS/MS method was successfully utilized for the determination of thrombin in clinical human plasma samples. One shortcoming of the method is its reduced efficiency in undiluted human plasma compared to the standard solution. Nevertheless, this new aptamer affinity-based SPME probe opens up the possibility of selective enrichment of a given targeted protein from complex sample either in vivo or ex vivo.