## 3 MIPs' Applications
### MIPs in Separation Techniques
More specifically peak broadening and tailing are both thermodynamic and kinetic characteristics. This phenomenon depends on the association constant and sample load, increasing for high values of association constant. Recent efforts to improve chromatographic efficiency have been done and include the replacement of non-covalent imprinting with the covalent one and the use of several strategies for obtaining uniformly sized spherical microspheres. MIPs have also been used as media for Capillary Electrochromatography (CEC). CEC is a hybrid separation technique that combines the stationary phase of LC with the electrosmotically driven mobile-phase transport of electrophoresis. MIP-based micro-columns for CEC are recently realized for separation of several compounds [73, 88, 89]. Solid Phase Extraction (SPE) is another important area of application of MIPs in analytical chemistry [21, 23, 90, 91, 92, 93]. MIP for Solid-phase extraction (MISPE) has been applied both in on-line and off-line procedures. MIPs particles, used as selective sorbent materials, can be packed in an HPLC precolumn for the on-line mode and in a cartridge between two fits for the off-line mode [14]. The on-line MISPE procedure, is directly coupled with specific analytical systems such as HPLC, minimizing samples manipulation and reducing the loss of analytes and the risk of contamination [92, 94]. Furthermore, this method considerably reduces the time for pretreatment of the samples. Despite these advantages, the off-line method is the most used for its simplicity. Moreover, more solvents can be handled without regard to their influence on the successive separation methods. The principle of MISPE is based on the same main four steps as classical SPE: conditioning of the sorbent, loading of the sample, washing away interferences and elution of the target analytes (Figure 6). In the loading step, the sample is percolated through the MIP sorbents. Generally, this solvent must have a polarity similar to that used in the polymerization process, since it increases the number of interactions between analyte and specific binding sites in the MIP sorbents [46]. MISPE was applied for extraction of several compounds in different sample matrices such as biological [54, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104], environmental samples [105, 106, 107, 108, 109, 110, 111] and also in food analysis [112, 113, 114, 115, 116, 117]. The first application of MISPE was made by [PERSON] in 1994 which prepared a selective extraction of pentamidine, a drug used for the treatment of AIDS-related disorders, in urine samples [95]. The pentamidine-imprinted materials were prepared by _in situ_ polymerization in a chromatographic column coupled with a simple HPLC system. Recently, some MISPE for vitamins and nucleoside, derivatives from biological matrices, were prepared. A highly selective MIP for 1-methyladenosine (1-MA), an urinary modified nucleoside which is used as cancer marker, has been prepared and used as sorbent material in an off-line SPE mode [54]. The polymer has been obtained by bulk polymerization using MAA as functional monomer and a mixture of acetonitrile/water (4:1, v/v) as porogen, to overcome the problems related to the imprinting of biological compounds. As can be seen from Figure 7, 1-MA has been selectively extracted by the MIP polymer after removing the interfering compounds present in the urine. Figure 6: The four steps of the MISPE process. Figure 7: Chromatograms corresponding to **(a)** human urine; **(b)** human urine spiked with 1-MA; and **(c)** elution solution after MISPE of spiked urine. Peak identification:1, 1-MA (Adapted from [54]). In another study [96] a selective molecularly imprinted polymer, obtained by bulk polymerization technique, was prepared as sorbent for solid phase extraction of nicotinamide. Moreover, the performances of the imprinted polymer have been compared to that of non-imprinted polymer and with conventional reversed-phase C18 performances and have shown better results (Table 1).