# A Compact Instrument for Temperature-Programming-Assisted Capillary-Nanoliquid Chromatography
## 3 Results
### Chromatographic Evaluation of the Fully Developed System  
The performance of the developed instrument was evaluated for the separation of PAH mixtures using packed (250 \(\upmu\)m o.d.) and alkylbenzene compounds using WCOT and PLOT open tubular columns (50 and 25 \(\upmu\)m i.d., respectively), showcasing its versatility for both conventional miniaturized LC and OTLC separations. Initial assessments were conducted using a C18-packed capillary column (14 cm \(\times\) 250 \(\upmu\)m \(\times\) 3.5 \(\upmu\)m). Figure 9 presents the chromatographic separation of a mixture of eight PAHs performed in both isothermal mode and under a temperature gradient. In part (a), the analyte was separated at a constant temperature of 30 \({}^{\circ}\)C. In part (b), separation was conducted with a temperature gradient, where the temperature was held at 30 \({}^{\circ}\)C for the initial 8 min and then linearly increased at a rate of 8 \({}^{\circ}\)C/min until reaching 85 \({}^{\circ}\)C, which was subsequently maintained for the next 30 min. For comparative analysis, parameters N, N/m, H, k, h, and E were calculated based on the last retained peak (dibenzo(a,h)anthracene), while Rs and \(\alpha\) were derived from the acenapthrene and fluorene peaks (Table 1). Retention times (t\({}_{\text{r}}\)) revealed complete separation under both conditions; however, the isothermal method required substantially longer total analysis time (40 min). Under temperature programming, the t\({}_{\text{r}}\) value for the last eluted peak was reduced by approximately 22 min, halving the analysis time. Figure 8: Graphs showing variation as a function of the squared retention volume for the (**a**) lab-made and (**b**) commercial instruments. Efficiency improvements were notable, with the temperature gradient increasing theoretical plates by about 34,000, equating to approximately 243,000 plates per meter. This represents a threefold increase in column efficiency. Temperature programming also \begin{table}
\begin{tabular}{l c c} \hline \hline
**Parameter** & **Isothermal** & **Temperature Programming** \\ \hline wb (50\%) Phenanthrene (min) & 0.817 & 0.2 \\ \hline N (Theoretical Plates) & 14,439 & 48,554 \\ \hline N/m (Plates per Meter) & 103,136 & 346,813 \\ \hline H (Height Equivalent to a  Theoretical Plate, \(\upmu\)m) & 9.7 & 2.88 \\ \hline R (Resolution) & 1.59 & 1.56 \\ \hline \(\alpha\) (Selectivity) & 1.35 & 1.25 \\ \hline k (Retention Factor) & 13.56 & 5.67 \\ \hline v (Linear Velocity) & 1.42 & 2.82 \\ \hline h (Reduced Plate Height) & 2.82 & 0.82 \\ \hline \(\mathcal{O}\) (Flow Resistance) & 1785 & 1488 \\ \hline E (Separation Impedance) & 13,700 & 1010 \\ \hline AP (Pressure Drop, bar) & 120 & 100 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Parameters extracted from the chromatograms in Figure 8, comparing isothermal and temperature-programmed separation modes. Figure 9: Separation of PAHs (50 mg/mL) on a packed C18 capillary column (14 cm length, 250 \(\upmu\)m i.d., 3.5 \(\upmu\)m particle size) at a flow rate of 5.0 \(\upmu\)L/min, using acetonitrile (70:30) as the mobile phase, under two conditions: (**a**) isothermal mode (no temperature programming) and (**b**) with temperature programming. Analytes: naphthalene (1), acenaphthylene (2), acenaphthene (3), fluorene (4), phenanthrene (5), anthracene (6), chrysene (7), and dibenzo(a,h)anthracene (8). impacted k and \(\alpha\), as both parameters decreased under the gradient. Further, temperature influenced retention time, which decreased as temperature rose, thereby increasing the linear velocity (v). A marked reduction in h, from 2.82 to 0.82, was also observed with TP application. Temperature additionally affected column pressure, with a backpressure reduction of approximately 20 bar. This influenced both flow resistance (\(\mathcal{O}\)) and separation impedance (E), with \(\mathcal{O}\) reducing to 291 and E decreasing by 12,690--an excellent result for coated capillary columns.