## Alternative Organic Solvents in RP-HPLC
### Ethanol
#### 2.1.2 Pharmaceutical Applications  
Green RP-HPLC methods using ethanol-based mobile phases have been widely reported for the analysis of drugs in pharmaceutical formulations [38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54], as well as in biological samples [55; 56] (Table 1). For example, a HPLC/diode-array detector (DAD) method using EtOH as the organic solvent in the mobile phase and an octadecyl-grafted silica column, was developed and validated by our team for the analysis of statins (i.e., pravastatin, fluvastatin, and atorvastatin in high concentrated EtOH/water solutions) [42]. The mobile phase was composed of an EtOH/aqueous solution of formic acid (pH 2.5, 25 mM) (50:50, \(v/v\)) at 40 \({}^{\circ}\)C. During method development, four columns with particle sizes between 1.9 \(\upmu\)m and 3 \(\upmu\)m were compared regarding efficiency using Van Deemter plots. For these acidic compounds, the benefit of keeping efficiency within a large range of flow rate, classically achieved through columns with particle sizes under 2 \(\upmu\)m, was not observed with ethanol-based mobile phases compared to acetonitrile ones (Figure 1). Such loss of efficiency with the flow rate may be a limit of the use of EtOH in some cases. [PERSON] et al. [45] developed and validated green HPLC methods, with mobile phases composed of EtOH/water mixtures at room temperature, for the analysis of coformulated pharmaceuticals treating gastrointestinal tract disorders: clidinium bromide/chlorodiazepepoxide hydrochloride,phenobarbitone/pipenzolate bromide, mebeverine hydrochloride/sulpiride, and chlorophenoxyoxamine hydrochloride/caffee/8-chlorotheophylline, either in their bulk powder or in their dosage forms. The developed methods were compared to the reported conventional HPLC methods and were found to be greener using the NEMI assessment [12]. More time-- and solvent-- saving was reported, hence these new methods can be used for routine analysis of the studied mixtures with a low impact on the environment. In another study, [PERSON] [38] developed a stability-indicating RP-HPLC method for the assay of prednisolone tablets using a mixture of EtOH/water (30:70, \(v/v\)) as the mobile phase. The separation was performed at 50 \({}^{\circ}\)C to overcome the increase in backpressure caused by the high viscosity of EtOH. Also, a green chromatographic method was developed and validated for the simultaneous determination of phenylephrine, paracetamol, and guaifenesin in their ternary pharmaceutical mixture using a mobile phase composed of EtOH and a phosphate buffer at pH 7.0 [47]. The separation was carried out on a monolithic column which allowed an efficient separation at higher flow rates, and consequently, a reduction in the analysis time. Lower quantities of solvent were consumed, and thus, a reduced amount of waste was produced. In another study, our team reported a green RP-HPLC method using a mobile phase composed of EtOH for stability studies of dextromethorphan [44]. The development was based on an innovative combination of green chemistry and Quality by Design (QbD) concepts to achieve a good quality of the method, while minimizing environmental impacts and analyst exposure. It is an example of how chemometrics can be used to aid in the development of green analytical chemistry. Experimental designs were used to identify parameters most affecting method performances, and to finally find the optimal operating conditions allowing the best chromatographic separation in terms of resolution between dextromethorphan and its degradation products, peak efficiencies, and solvent consumption. A response surface methodology allowed determining the Design Space (DS) (method operable design region), which was validated experimentally to prove method robustness inside. This method was validated using the concept  
Figure 1: Comparison of Van Deemter curves of atorvastatintian obtained using ethanol or acetonitrile in mobile phases.