Variables 
Levels 

Low 
Medium 
High 
Independent variables (factors) 



X_{1}; Flow rate: mL/min 
0.6 
0.8 
1.0 
X_{2}; Injection volume: µL 
10 
15 
20 
X_{3}; Detection wavelength: nm 
265 
270 
275 
X_{4}; Initial gradient acetonitrile ratio: % v/v 
60 
70 
80 
X_{5}; Acetonitrile ratio at four min of gradient run: % v/v 
85 
90 
95 
Coded levels of levels of X_{1}, X_{2}, X_{3}, X_{4} and X_{5} 
1 
0 
+1 
Dependent variables (responses) 
Y_{1}; Peak area of STP: mAU.s 
Y_{2}; Peak area of HI443: mAU.s 
Y_{3}; USP Tailing of STP 
Y_{4}; USP Tailing of HI443 