## 3 Analytical Instrumentation
### Chromatography in Water Analysis
UPLC uses small diameter particles (typically 1.7 \(\upmu\)m) in the stationary phase and short columns, which allow higher pressures and, ultimately, narrower LC peaks (5-10 s wide). In addition to providing narrow peaks and improved chromatographic separations, UPLC dramatically shortens the analysis times, often to 10 min. or less [49; 86]. Therefore, it provides several advantageous characteristics, and the best ones are: employs less sample volume, provides faster analysis, less mobile phase consumption, higher resolution of chromatographic peaks, better detectability, and higher signal/noise ratio, which, in turn, decreases the limit of quantification (LOQ) [170; 171; 172]. Although the assortment of columns currently available for HPLC and UPLC is wide and varied, most methods being applied to PhACs analysis have been based primarily on the use of C18 reverse-phase (RP) columns (Table 1). However, columns C8 [3; 90; 104] and C16 [150] have also been used. The C16 is an RP-amide column used for the same separations as a C18, but shows higher wettability in high aqueous mobile phases. It is much more retentive for those PhACs that can interact by hydrophobic interactions and H-bonding with the amide group. Therefore, it is the right column for acidic PhACs. The 1.7 \(\upmu\)m and 1.8 \(\upmu\)m UPLC BEH (Ethylene Bridged Hybrid) columns are the most used in UPLC. These columns allow a wide pH range (pH 1-12) due to the intrinsic chemical stability of hybrid particle technology. Therefore, they have been applied to the analysis of a great diversity of PhACs [37; 45; 94; 101; 173]. The UPLC HSS T3 columns have also been employed for PhACs analysis by UPLC in waters matrices. The HSS (High Strength Silica) particle is 100% silica ideally suited for the enhanced retention of polar compounds and metabolites by reversed-phase LC. This low-ligand density C18 column enables analytes to access the material's pore structure more readily, providing a balanced retention of polar and hydrophobic molecules without the need for ion-pair reagents. The compatibility with 100% aqueous mobile phase and analysis of polar and non-polar compounds is an excellent advantage of these columns for PhACs analysis in water matrices [37; 78; 83; 174; 175]. The HPLC/UPLC with UV [123; 125; 127; 176; 177], DAD [111; 114; 150; 161; 162] and FLD [124] detection was used in the analysis of PhACs, but, in most applications, it is coupled with tandem mass spectrometry [37; 45; 98; 101; 178; 179]. Mass spectrometry characteristics have raised it to an excellent position among analytical methods, mainly due to its sensitivity and detection limits. Regarding water matrices, optical detectors in LC methods for the analysis of PhACs are limited to wastewater, because its threshold limits are insufficient for measuring these target compounds at trace levels in groundwater and drinking water, even in surface waters. The enrichment factor of sample preparation method, even the large one used in the SPE technique (1000), is not enough to overcome their lower sensitivity. For example, the limits of quantification (LOQs) of 12 PhACs from four therapeutic classes by SPE-HPLC-UV-FLD (1000 mL of sample) ranged between 10-800 ng/L in surface waters, 10-850 ng/L in wastewater effluents, and 30-1100 ng/L in ranged between 10-800 ng/L in surface waters, 10-850 ng/L in wastewater effluents, and 30-1100 ng/L in wastewater influents [177]. The analysis of 90 PhACs of thirteen therapeutic classes by SPE-HPLC-MS/MS ranged between 0.06-196 ng/L in surface waters and 0.07-78 ng/L in wastewater effluents [46]. When considering the PhAC with the lowest LOQ in both techniques, the SPE-HPLC-UV-FLD method has a sensitivity approximately 150 times lower than that of the HPLC-MS/MS method, even using twice the sample volume. The UPLC coupled to mass spectrometry has undoubtedly seen the most significant developments (almost at an exponential level) for applications in the field of research, but, above all, in routine analysis.