# A Compact Instrument for Temperature-Programming-Assisted Capillary-Nanoliquid Chromatography
## 2 Materials and Methods
### Chromatographic Columns  
Laboratory-prepared C18-packed capillary columns were manufactured in polyether ketone (PEEK) tubes of 0.25 \(\upmu\)m i.d., using a particle suspension technique. First, a suspension containing 16 mg of 3.5 \(\upmu\)m C18 particles (Cromatorex, Nagoya-shi, Japan) and 0.7 mL of a THF/IPA solution (1:6 \(v/v\)) was stirred and transferred into a packing column connected to a Shimadzu pumping system. Methanol was pumped at 200 bar for one hour to ensure adequate packing. After this, the pump was turned off, and the column was left in place for an additional hour to ensure complete depressurization. The column was then washed with MeCN for 30 min.  
A wall-coated open tubular column (WCOT) was prepared using fused silica tubing (50 \(\upmu\)m i.d., 375 \(\upmu\)m o.d., 2.30 m length) from Polymicro Technologies (Phoenix, AZ, USA). The tubing was pretreated by flushing it with a 2% \(v/v\) hydrochloric acid (HCl) solution (Tedia, Fairfield, CT, USA) under a nitrogen flow at 80 bar for 5 min. It was then placed in an oven at 220 \({}^{\circ}\)C for 3 h. Following this, the tubing was washed with water and dichloromethane, 1.5 mL each, and dried with a nitrogen stream--a dehydration step involved heating the column at 200 \({}^{\circ}\)C for 2 h under a continuous nitrogen flow. Silanization was achieved using hexamethyldisilazane (HMDS) from Sigma-Aldrich (St. Louis, MO, USA), with the tubing heated at 290 \({}^{\circ}\)C for 6 h. A stationary phase coating (50% methyl and 50% phenyl polysiloxane) was applied by percolating a 2% \(w/v\) solution of the stationary phase through the column for 30 min. The column was then heated at 40 \({}^{\circ}\)C for 15 min. Film immobilization (0.25 \(\upmu\)m thickness) was achieved by percolating azo-tert-butane (ATB) through the column for 5 min and heating at 220 \({}^{\circ}\)C for 1 h. This immobilization process was repeated three times to ensure stable film adherence.  
A porous layer open tubular column (PLOT), a 5 m long tube with a 25 \(\upmu\)m internal diameter, was also prepared using a polystyrene divinylbenzene (PS-DVB) stationary phase. Tube pretreatment began by filling it with a 1 mol/L NaOH solution, followed by placement in an HPLC oven at 60 \({}^{\circ}\)C for 20 h. The tube was subsequently washed with H\({}_{2}\)O and MeCN for 30 min each and dried with N\({}_{2}\) gas for 30 min. For silanization, the tube was treated with a solution of 40 \(\upmu\)L \(\upgamma\)-MAPS and 10 mL of 6 mol/L acetic acid. The capillaries were filled with this solution, sealed, and heated in an HPLC oven at 60 \({}^{\circ}\)C for 20 h. The tubes were rewashed with H\({}_{2}\)O and ACN for 30 min each and dried with N\({}_{2}\) gas. The polymerization solution was prepared by mixing 5 mg of AIBN, 200 \(\upmu\)L of styrene, 200 \(\upmu\)L of divinyl benzene, and 600 \(\upmu\)L of ethanol in an Eppendorf tube followed by 5 min of ultrasonic sonication. After filling, the tubes were sealed and placed in a thermal bath at 74 \({}^{\circ}\)C for polymerization. Finally, the PS-DVB PLOT column was washed with H\({}_{2}\)O and ACN for 30 min each and dried with N\({}_{2}\) gas for at least 1 h.