# A Compact Instrument for Temperature-Programming-Assisted Capillary-Nanoliquid Chromatography
## 3 Results
### Chromatographic Evaluation of the Fully Developed System
Reduced parameter analysis confirmed high column performance, further enhanced by temperature programming. Temperature programming was also applied to WCOT and PLOT open tubular columns, demonstrating its efficacy in reducing analysis time and enhancing peak intensity and column performance. For this purpose, a WCOT OTLC-4 column (50 \(\upmu\)m i.d.) and an OT-PS-DVB polymer column (25 \(\upmu\)m i.d.) were used to separate a mixture of five alkylbenzenes and uracil as a dead time marker. For the separations on the WCOT column (Figure 10), a mobile phase of \(\mathrm{ACN}/\mathrm{H}_{2}\mathrm{O}\) (60:40 \(v/v\)) was employed at a flow rate of 5 \(\upmu\)L/min, with analyte standards at a concentration of 1.4 \(\upmu\)L/mL, and detection at 210 nm. The isothermal run was maintained at 30 \({}^{\circ}\)C, while for temperature programming, the oven was set to increase from 30 \({}^{\circ}\)C to 100 \({}^{\circ}\)C over 14 min at a rate of 5 \({}^{\circ}\)C/min. When using the PLOT column, \(\mathrm{ACN}/\mathrm{H}_{2}\mathrm{O}\) (40:60 \(v/v\)) was used as the mobile phase at a flow rate of 3 \(\upmu\)L/min, with detection at 210 nm. The isothermal run was also conducted at 30 \({}^{\circ}\)C, and for temperature programming, the column temperature was held at 30 \({}^{\circ}\)C before increasing to 80 \({}^{\circ}\)C over 10 min at 5 \({}^{\circ}\)C/min. The resulting chromatograms, showing alkylbenzene separations in both isothermal and temperature-programmed modes, are presented in Figure 11.  
Figure 10: Separating alkylbenzene compounds using a WCOT column (5 m length, 50 \(\upmu\)m i.d. ): (**a**) without temperature programming and (**b**) with temperature programming. Peaks: uracil (1), Toluene (2), ethylbenzene (3), butylbenzene (4), pentylbenzene (5), and heptylbenzene (6). As illustrated, temperature programming significantly reduced the analysis time for both open tubular columns and generally increased peak intensities. However, the gain in signal was less pronounced than with packed columns. In isothermal mode, the WCOT column could not resolve the last eluted peak; however, temperature programming allowed for well-defined shapes for all six alkylbenzene peaks. Additionally, retention time (t\({}_{\text{r}}\)) reduction was accompanied by narrower peak widths, increasing the WCOT column efficiency from 972 to 2442 theoretical plates for the heptylbenzene peak--a 2.5-fold enhancement, indicating the value of temperature programming in open tubular liquid chromatography. The same trend was observed with the PLOT columns, where retention time reduction and peak narrowing improved signal intensity for the later eluting analytes. For example, efficiency for the pentylbenzene peak doubled with temperature programming compared to isothermal conditions. It is essential to highlight that the detectability observed with the PLOT column was approximately five times higher than that of the WCOT column. This difference can be attributed to variations in on-column dilution during the elution process, which is inversely proportional to the square of the column's radius and length [2]. In this case, the PLOT column, with nearly half the radius and length of the WCOT column, facilitated the elution of more concentrated analyte bands. This explains the differences in noise levels and detectability observed in Figures 9 and 10. The success of temperature programming in capillary LC demonstrates its potential as an alternative to solvent gradient methods, reducing analysis time and enhancing column performance while simplifying instrumentation by eliminating the need for multiple pumps and solvent gradient equipment.