## 4 Molecular Imprinted Polymer in Solid Phase Extraction (MISPE)
### Online Modes  
In contrast to the offline mode, as shown in Figure 15, the online mode provides for automatic sample loading, interference washing, analyte elution, separation, and detection by an analytical equipment. In a typical process, a tiny pre-column filled with molecularly imprinted polymers is placed in the loop of a six-port injection valve. The sample is placed into the analytical column, interferences are removed, and the analytes are eluted and separated in the analytical column before being evaluated by the detector [95]. The following is a schematic diagram of the MISPE steps in online mode:  
In online MISPE mode, the left pump conducts the autosampler procedure. The sample is then discarded after passing through the online cartridge. The right pump inflates the mobile phase into the analytical column, which is subsequently examined by the instrument. The left pump works in tandem with the correct pump during the eluting process, as shown in Figure 15b. The difference between the loading and washing processes is that the left pump's sample does not go through an online cartridge, instead it goes through the right pump's mobile phase. The left pump transports the mobile phase, while the auto sampler transports the sample and mobile phase to the loops of port injection valves 2 and 3, then into the online SPE cartridge (containing the analyte), and the solvent to the loop of port injection valve number 1, before the stream is diverted to waste (see Figure 15a). The washing solvent is then transported to ports 2 and 3 via the left pump, entering the online SPE cartridge to wash the sample in the online SPE cartridge (Figure 15a). Next, the interferences stream is discharged, but the analyte remains trapped in the online cartridge. Figure 15a depicts the optimal pump that is used in the eluting process. The right pump transports the eluting solvent through ports 5 and 4 before passing it through the analytical column to elute the analyte for mass spectrometry (MS/MS) analysis. During the eluting stage, the correct pump in Figure 15b transports the eluting solvent into the online SPE cartridge through ports 5 and 6, which carries the analytes from the online cartridge to ports 3 and 4 for MS/MS analysis. Figure 15b also depicts the process of the analyte being trapped in the online SPE cartridge and the stream being diverted to waste early in the loading step for the left pump. If the mobile phase is inadequate to elute the analytes from the MIP pre-column, another eluting solvent can be utilized. The eluted analytes then pass through the injection loop before being injected into the chromatographic set-up, where the mobile phase carries them away [90; 98]. Figure 15: Molecular imprinted solid-phase extraction (MISPE) steps in online mode (**a**) Sample Loading/Washing; (**b**) Sample Eluting [95]. The online method has certain significant advantages over the offline approach, including solvent conservation and low cost. In some situations, the analytes are collected in a minimal solvent volume after loading the full sample extract through the MIP's bed, resulting in a superior concentration factor. The MSPE online mode is most suited to multi-analyte determinations, where the MIP detects numerous structural analogues [90; 99]. As a result, it is not as popular as the other modes. As an example of analytes analyzed using the online SPE method, [PERSON] et al. [100] used simazine as a template in MIPs to extract triazines from humic acid (SPE). Online trace enrichment of water samples containing 20 ppm humic acid was proven to be successful using MIPs. Because of the MIP selectivity, large volumes of water samples containing humic acid (20 ppm) can be extracted with little influence from the matrix on the chromatogram, thus, high sample-enrichment factors could be obtained. On the other hand, sample matrixes like apple extracts or urine were retained on the MIP to some extent, limiting the amount of samples that could be extracted but still resulting in sample enrichment [100].