Procedure (Note 1):

1. 1.  Turn on the HPLC instrument and the software as per the manufacturer's instructions.

2.  Wash the inlet filter of the tube connected to pump A (labeled as line ‘A') with solvent A and place the filter in the bottle having solvent A.

3.  Similarly, wash the inlet filter of the tube connected to pump B (labeled as line ‘B') with solvent B and place the filter in the bottle having solvent B.

4.  Open the purge valve. Opening the purge valve causes the solvent to bypass the column and travel directly to the waste liquid container.

5.  Set the pump at 100% solvent A with a flow rate of 1 ml/min .

6.  Allow the solvent A to run for 10 minutes, ensure that there are no bubbles in the solvent line.

7.  Set the pump at 100% solvent B keeping the flow rate at 1 ml/min .

8.  Allow the solvent B to run for 10 minutes, ensure that there are no bubbles in the solvent line.

9.  Stop the flow by setting a flow rate of 0 ml/min .

10.  Close the purge valve.

11.  Set the pump at 50% solvent A with a flow rate of 0.5 ml/min. Now the eluant will enter the injector and come out of its outlet. You may see some bubbles coming out if injection loop were dry.

12.  Allow the eluant to run till the eluant starts flowing without any air bubble.

13.  Stop the flow by setting a flow rate of 0 ml/min.

14.  Open both the caps of the column and connect it with the injector outlet in the proper orientation (Reversed-phase columns come with flow direction indicators).

15.  Set the pump at 100% solvent B keeping the flow rate at 0.5 ml/min.

16.  When the solvent starts coming out of the column, attach the column with the detector inlet.

17.  Wash the column with 100% solvent B at a flow rate of 0.5 ml/min for at least 20 minutes.

18. Meanwhile, wash the injection loop with solvent B at least 5 times followed by washing with solvent A at least three times.

19.  Set the pump at 100% solvent A with a flow rate of 0.5 ml/min and allow column equilibration.

20.  Set up the instrument parameters:

a.  Set the flow rate to 0.5 ml/min for the HPLC runs.

b.  Select the UV absorption for detection.

c.  Set the wavelength to 214 nm. If instrument can record absorbance at multiple wavelengths, as is the case with Photodiode Array Detectors, select two more wavelengths: 254 nm and 280 nm.

d.  Set up the gradient as shown in figure 26.3.