Table 1.
Optimization of HPLC-UV conditions for three analytes.
| Chromatographic factor | Compared parameter | Optimized conditions | Main advantages |
|---|---|---|---|
| Column | Phenomenex Hydro_RP (4.6 mm × 250 mm, 4 μm), Kinetex-C18 (4.6 mm × 100 mm, 2.6 μm) |
Kinetex-C18 | Improved column efficiency, speed, separation, and sensitivity |
|
| |||
| λ UV | 203~210 nm for CA and AU, 235~240 nm for GPA | 210 nm for CA and AU; 240 nm for GPA | No difference in 203~210 nm; less miscellaneous peaks and stabler baseline at λ 240 nm |
|
| |||
| Mobile phase | Methanol-H2O, ACN-H2O | ACN-H2O | Better peak shape and separation, stabler baseline |
| Adding or not adding of formic acid in H2O | (0.1% formic acid) | Better peak shape and separation for GPA; no obvious influence on CA and AU | |
| Linear gradient or isocratic | Isocratic | Smooth baseline, better separation, less miscellaneous peaks | |
|
| |||
| ACN : H2O (0.1% FA) | 1 : 99, 2 : 98, 3 : 97, 5 : 95, 7 : 93, 10 : 90 | 5 : 95 | Shorter elution time; however, CA overlapped with solvent peak when ACN was >5%; longer elution time for AU and GPA when ACN was less than 5% |