The goal was to locate the optimum electrophoretic conditions that allow the minimal analysis time for the 5-HMF determination. A full factorial design (11 experiments) containing three selected factors, was chosen as a 32

full factorial design with three trials at the central point. The factors and their “low” (−) and “high” (+) levels are summarised in Table 2. The individual runs of the design were selleck carried out in a randomised sequence. Randomisation offers some assurance that the uncontrolled variation of factors, other than those being studied, will not influence the estimation (Micke, Fujiya, Tonin, Costa, & Tavares, 2006). The replicate measurements were stable and the capillary was well-equilibrated after changing to new electrophoretic conditions. Multiple regression enabled the mathematical relationship between the responses and the independent variables to be determined. The width and the migration time of Palbociclib ic50 5-HMF and caffeine were computed as a function of the electrolyte composition according to the following empirical equation: equation(1) tiorRw1/2=constant+a[STB]+b[SDS]+c[MeOH]where, t is the migration time of the analyte

i and w is the width of the analyte peak. The equations were solved numerically by means of the Solver algorithm (Microsoft® Excel 2007) and the coefficients are organised in Table 3. The experimental results very obtained from the factorial design were used for modelling the width and migration time of the peaks. With these data, it was possible to estimate the response provided by Eq. (2): equation(2) Resp.=Rtcafwhere R is the resolution between 5-HMF and caffeine, and tcaf is the migration

time of caffeine (IS), the last peak on the electropherogram. The resolution (R) was calculated using Eq. (3), where t1 and t2 are the migration times, and w1 and w2 the baseline widths of the HMF and caffeine peaks, respectively. equation(3) R=t2-t10.5(w1+w2) The response function (Eq. (2) was calculated for the entire dataset, and a response surface was generated (data not shown) indicating the optimum conditions for separation with the electrolyte composed of 5 mmol L−1 STB and 120 mmol L−1 SDS, at pH 9.3. The corresponding electropherogram of a solution of 5-HMF and the caffeine standards under optimised conditions is shown in Fig. 1. The analysis time was successfully reduced using the short-end-injection mode (Ldet 8.5 cm) and a high electrical field (468.8 V/cm). A baseline separation of 5-HMF and caffeine (IS) was achieved, with high resolution, within 42 s. This separation time is considerably shorter than that of other CE methods reported in the literature. The online acquired UV spectra are depicted in the insert of Fig. 1.