• 25.  The HPLC run will be complete after 65 minutes (Figure 26.3).

26.  As the equilibration step for the next run is already incorporated in the gradient program, the column is ready for next injection.

27.  If the ‘Blank' run is clean, inject your sample as discussed in the subsequent steps. (If the blank run is not clean, wash the column with 100% B at a flow rate of 1 ml/min for 30 minutes and wash injection loop 10 times with solvent B followed by 3 times washing with solvent A. Repeat the blank run).

28.  Include the sample information and the file name for the next run.

29.  Turn the injector knob to ‘Load' mode, inject 100 μl of the protein/peptide sample, and turn the knob to ‘Inject mode'.

30.  Monitor the HPLC run and collect the peaks, if the protein/peptide is to be analyzed/used further.

31.  After the completion of HPLC run, set the flow rate to 0 ml/min .

32.  Once the instrument displays 0 ml/min flow rate, take out line B from the solvent bottle.

33.  Rinse the inlet filter of line B with methanol and put it in methanol bottle.

34.  Open the purge valve.

35.  Set the pump at 100% solvent B (which is methanol now) with a flow rate of 1 ml/min .

36.  Allow the methanol to run for 10 minutes.

37. Close the purge valve.

38.  Set the pump at 100% B with a flow rate of 1 ml/min and let it run for 20 minutes.

39.  Set the flow rate to 0 ml/min .

40.  Once the instrument displays zero pressure, switch ‘off' the instrument as per manufacturer's instructions.

41.  Remove the column, cap both the ends and store it at room temperature till further use.

42.  Connect the outlet of the injector and the inlet of the detector with an adapter provided with the instrument; this slows down drying of the solvent lines.

Results and analysis:

1. 1.  Go to ‘Data Analysis' after the run is complete; in modern instruments, the software automatically calculates the area under the peaks.

2.  Calculate the Percentage purity of the peaks as follows:

1. 3.  The peak that corresponds to the protein/peptide of interest is identified by determining the mass of the collected fractions using mass spectrometry.

Notes:

1. 1. The exact procedure may vary from instrument to instrument but the procedure discussed here should give the user an idea how to go ahead for performing reversed-phase chromatography on any HPLC instrument.