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A previous cLC method, involving large injection volumes (20 μL) and on-column focusing techniques, provided a suitable sensitivity using an Inertsil? C8 capillary column, DAD and gradient elution. This method showed that composition of the focusing solution, mobile phase and pH have remarkable effects on HA separation. In order to improve this separation, an isocratic cLC method compatible with MS detection was developed and the influence of several chromatographic factors on separation quality evaluated.
Different reversed-phase packings such as Luna? C8, Luna? C18, Synergi? Max-RP (C12) and Synergi Fusion (C18 and polar embedded group) were tested using UV-DAD detection. These columns were chosen due to their high efficiencies and bonded phase surface coverage. Considering the different particle sizes, flow rate was set at 15 μL min?1 for both Synergi Fusion? and Synergi? Max-RP, 12 and 9 μL min?1 for Luna? C8 and Luna? C18 columns respectively.
Chromatographic conditions were optimized using experimental design methodologies such as central composite design. Factors and ranges selected for the optimization of mobile phase composition were ACN (11.6–28.4%), ammonium acetate concentration (13.2–46.8 mM) and buffer pH (3.6–4.4). Response variables were expressed in terms of resolution, calculated at baseline, between the worst resolved peak pair NH–H (Rs,min), and retention time of the last eluting peak (tend). From the obtained equations, it could be concluded that Rs,min was significantly affected by both ACN percentage and pH while tend only by ACN. In addition, the ammonium acetate concentration had no significant effect on separation.
Chromatographic separation efficiency was optimized for Rs,min ≥ 2 and minimum tend value. Luna? C8 capillary column provided poor resolution. Luna? C18 and Synergi? Max-RP columns provided similar Rs,min to Synergi? Fusion but higher efficiencies. However, Synergi? Max-RP column provided poor peak symmetry. Therefore, Luna? C18 column was selected for further optimization with the quadrupole MS detector.
Taking into account the instrumental limitations of the MS capillary nebulizer, the ammonium acetate concentration in the mobile phase was fixed to 5 mM. Chromatographic separation was optimized using multifactorial design. Factors and ranges selected were ACN (12–18%) and pH (3.6–4.1). For the injection, buffered focusing solutions with 5% MeOH were used.
Table 1 includes the values of the experimental responses (Rs,min and tend), which were fitted into the following normalized polynomial equations:
(1)Rs,min=1.74?0.51pH?1.35ACN?0.06pH2+0.39pH ACN+0.40ACN2
(2)tend=12.18+0.18pH?11.02ACN+0.76pH2?0.94pH ACN+4.83 ACN2
Determination coefficients were 0.988 and 0.992 for Rs,min and tend respectively, showing the reliability of the equations. In the studied domain, both ACN percentage and pH affected significantly to Rs,min (p values 0.0000 and 0.0002 respectively) (Eq. (1)) while ACN (%) affected only tend (p value 0.0000 and 0.0005 for the single and quadratic term respectively) (Eq. (2)). As can be expected, the interactions between factors (ACN-pH) were significant only for Rs,min response (p = 0.0033). In Fig. 1 it can be observed that the ACN percentage decrease in the mobile phase increases Rs,min and tend. However, when pH decreases, Rs,min increases and tend does not change significantly.
The chromatographic separation efficiency was optimized by maximizing Rs,min and minimizing tend values which maximized the desirability function over the selected region. The maximum desirability function yielded a tend = 13.6 min and Rs,min = 2.2 at pH 3.6 and 14.5% ACN. The estimated response surfaces predicted that Rs,min > 2 and tend in the range 12.5–15.0 min could be obtained at pH 3.6–3.7 and 13–15% ACN. As a compromise, pH 3.6 and 13% ACN were selected as optimum values. Under these conditions tend = 15.0 min and Rs,min = 2.3 were expected and experimentally assessed. Finally, ammonium formate 5 mM was also tested and selected to avoid problems into the capillary MS nebulizer.

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