Ionic liquids (ILs) and monolithic materials are the emerging trends in chromatographic separations. In the present investigation 3-methylimidazolium cation-based ionic liquids were evaluated as mobile phase additives to check their compatibility with monolithic columns for separation of antiretroviral drugs by RP-HPLC. Effect of their concentration, polarity and viscosity on peak asymmetry and resolution was studied. Multiple interactions of ILs with analytes as well as stationary phase were found to be beneficial in optimizing the separation of antiretrovirals.

The unique properties of ionic liquids such as non volatility, non flammability, excellent chemical and thermal stability, make them an environmentally attractive alternative to conventional organic solvents. ILs are usually composed of relatively large organic cations and inorganic/organic anions [1]. These are widely used in analytical separations. The recent breakthrough in LC column technology was the development of monolithics. These were conceptually designed and developed to reduce analysis time through low back pressures. These materials allow not only high flow rates but also fast mass transfer kinetics. However, their plate heights are lower than those of conventional columns which make them less advantageous for basic compounds. It was also not clear that the poor peak shapes for strong bases were due to construction morphology of monoliths or residual silanol groups [2, 3]. Basic compounds are difficult to separate on particulate materials by RPLC due to the interactions between cationic sites of the compounds with the anionic silanols of the stationary phases. As a result peak tailing and long retention times of the basic compounds were observed.

Such problems are generally overcome by (i) end capping the free silanol groups or (ii) adding mobile phase additives like triethylamine to suppress the residual silanol groups [4]. Recently, ILs were proposed as possible candidates to suppress the residual silanol activity of RPLC columns. A few articles reported on the effect of ILs on particulate C18 material but not on porous C18 material (monolith) in separation of drugs and pharmaceuticals [5].

The morphology, the average size of their pores, and size distributions of these pores are different compared to conventional particulate columns (fig. 1). Hence, the compatibility of ILs with monolithic material is still ambiguous. In addition, different types of ILs are available with wide ranges of polarity and viscosity. The problem in selecting ILs as possible organic additives in RPLC is their very high viscosity. The most prominent imidazolium based ILs and antiretroviral drugs were selected as testing probes in the present study (Table 1).

Material and Methods
Chromolith Flash, RP-18e column (25 x 0.46 mm) using water (pH: 4.0 adjusted with acetic acid) (A) and methanol (B) v/v was used as mobile phase. Fig. 2 represents the chromatographic separation of eight ARVs in gradient elution: 0.01 min-5% B, 4.0 min-25% B, 6.0 min-70% B, 11.0 min- 25% B, 12.0 min- 20.0% B, 13.0 min - 5% B, 17.0 min- 5% B at a flow rate of 1.2 mL/min and detection at 254 nm.

Results and Discussion
To understand the effect of ILs, it is important to know their interactions with stationary phases as well as solutes in the mobile phase. Due to the dual nature of ILs, the analytes are retained by a combination of hydrophobic, ion pairing, ion exchange and other electrostatic interactions with the stationary phase and with ionic mobile phases (fig. 3). The equilibrium depends upon the dominating power of any one or sum of the interactions mentioned above. The retention of basic drugs in ion pair chromatographic separation in presence of small hydrophilic anionic additives/anionic part of ILs follows the order H₂PO₄^-<HCOO^-<CH₃SO₃^-<Cl-<NO₃^-<CF₃COO^-<BF₄-<ClO₄^-<PF₆^- [6]. Typically, both basic and acidic ion pairing reagents are required to reduce the peak asymmetry of both acidic and basic analytes. However, only one IL was found to be enough to serve both purposes, due to its dual nature.

Effect of Concentration
The addition of ILs to the mobile phase generally improves the peak shapes due to ion pairing between ILs and solutes of cation in nature. From Fig. 2a, it is clear that the peaks of nevirapine, tenofovir and efavirenz experienced tailing in absence of ILs. But their tailings were completely disappeared when BMITB and EMIIMS were used as ILs (Fig. 2b). Additionally, the solutes experienced less asymmetry with BMITB than BMIB, because the BF₄^- ions have a chaotropic character allowing them to associate with cationic solutes, whereas Br^- ions are kosmotrope and unable to make such ion pairing with cationic solutes. Unfortunately, a highly tailed and drift in base line separation was found in presence of HMIC and BMIOS ILs. It could be due to the high viscosity and a less polar nature of ILs. These two ILs are partly soluble in aqueous media and freely soluble in presence of organic modifiers.