6 mm, i.d, 5 μm) at 40 °C. The mobile phase was acetonitrile–water with 0.5% phosphoric acid in gradient: Acetonitrile: 0–8 min, 35–50%; 8–14 min, 50–60%. The flow rate was 1.0 mL/min and the injection volume was 10 μL. The CPs was monitored at 215 nm by a photodiode array detector. In IL-DLLME, extraction agent IL was an important factor to affect the extraction efficiency. Most of the ILs Apoptosis Compound Library purchase are composed of cations (e.g., imidazole, pyrrolidine, pyridine) with inorganic anions (e.g., Cl−, PF6−, BF4−). The composition of different cations and anions ion could form 1018 kinds of ILs, thus how to choose the ionic liquid is of difficulty. This paper followed the following principles of IL selection

(1) in situ IL-DLLME is organised on the basis of two elements: hydrophilic IL (anions e.g., Cl−,

Br−, BF4−) and anion-exchange reagent (e.g., NaPF6, LiNTf2); (2) ILs must be liquid under the experimental conditions; (3) formed hydrophobic ILs have a greater density and smaller viscosity than water for easy separation of sedimentary phase from aqueous sample. (4) Anion exchange reaction does not affect extraction of the target substance. Three kinds of hydrophilic ILs including [C4MIM][BF4], [C4MIM][Cl] and [C4MIM][Br] were tested separately to enrich CPs by reaction in-situ with LiNTf2 forming hydrophobic [C4MIM][NTf2] as extraction agent, this website which has greater density and smaller viscosity. As can be seen in the Fig. 1, the CPs enrichment recoveries were higher using hydrophilic [C4MIM][BF4] than [C4MIM][Cl] and [C4MIM][Br], and also higher than hydrophobic [C4MIM][PF6] as direct extraction agent. Thus, [C4MIM][BF4] was selected as the suitable ILs. To investigate the effect of the molar ratio of hydrophilic IL to anion-exchange

reagent on the extraction efficiency of CPs, different volume of LiNTf2 aqueous solution (0.51 g/mL) were tested when [C4MIM][BF4] was fixed at 80 μL. Fig. 2A shows obviously that when molar ratio of [C4MIM][BF4] to LiNTf2 reached 1:1, corresponding to the point of 240 μL LiNTf2 aqueous solution, the recoveries of CPs reached D-malate dehydrogenase the maximum. Then keeping the 1:1 molar ratio, different volume of [C4MIM][BF4] and LiNTf2 aqueous solution were tested to form different volume of hydrophobic [C4MIM][NTf2] to investigate its effect on the extraction efficiency of CPs. As seen in Fig. 2B, the enrichment recoveries of CPs increased with the increase of [C4MIM][BF4] volume from 40 μL up to 100 μL probably due to the improvement of mass transfer effect. However, further increase of the volume resulted in a slight decrease of the extraction recoveries due to the dilution effect. Thus, the following experiments were carried out by using 100 μL [C4MIM][BF4] and 300 μL LiNTf2 aqueous solution. The pH of the solution is an important factor affecting the extraction efficiency, especially when extracts is weak acidic or weak alkaline. The six studied CPs are weak acids with pKa values in the range of 6.0–9.4 (De Morais et al.