Recently, the multilayer coil was introduced in type-I CCC system, which showed the excellent peak resolution. Its performance was further improved by intermittently pressed tubing. In the present study, the whole plain tubing was pressed to make the flatten tubing. Then, it was twisted along its axis to form the flat-twisted tubing. The performance of these two tubing geometries was tested in type-I CCC system with three solvent systems of different polarities each with the suitable set of test samples of DNP-amino acids, dipeptides and proteins. The results showed that the retention of stationary phase was increased with the decreased flow rates and the increased polarity of solvent systems. In DNP-amino acid separation, flatten tubing produced the best peak resolution among all tubing geometries, where the resolution of three peaks was 2.17 and 2.30, respectively. In dipeptide separation, the result of flat-twisted tubing was good, but slightly worse than that of intermittently pressed tubing due to the lower retention of stationary phase. In protein separation, the resolution of intermittently pressed tubing was the best. The present study provided more options and ideas for type-I CCC application and development.