C18 HPLC Column Washing Procedure
Purpose
To describe the procedure for HPLC column washing.
Scope
This procedure is applicable for procedure for HPLC column washing
Procedure: C18 HPLC Column Washing Procedure
· Reverse phase HPLC column washing procedure (Isocratic analysis method):
· Flush the Column with 95 % water and 5 % Methanol for 15 minutes.
· Flush the column with 5 % water and 95 % Methanol for 15 minutes.
· Flush the column with 5 % water and 95 % Acetonitrile for 15 minutes.
· Run the Gradient Program through HPLC column flow rate 1.0 ml/min for 45 minutes.
· Washing method parameter are as described below;
|
Sr. No. |
System parameter |
Parameter |
value |
|
1. |
Data Acquisition |
LC Stop time |
45.01 |
|
2. |
Pump |
Mode |
Low pressure gradient |
|
|
Flow |
1.0000 ml/min |
|
|
Line C Conc. |
5 % |
||
|
Line D Conc. |
95 % |
||
|
Pressure Max |
449 kgf/cm2 |
||
|
3. |
Auto sampler |
Auto sampler |
Enable |
|
|
Cooler temperature |
25°C |
|
|
4. |
Oven |
Oven Temperature |
40°C |
|
5. |
Detector A |
Lamp |
Off |
Gradient Programming is as mentioned below. C18 HPLC Column Washing Procedure
|
Time |
Module |
Command |
value |
|
0.01 |
Pumps |
Solvent B Conc. |
0 |
|
0.01 |
Pumps |
Solvent C Conc. |
5 |
|
0.01 |
Pumps |
Solvent D Conc. |
95 |
|
15.00 |
Pumps |
Solvent B Conc. |
0 |
|
15.00 |
Pumps |
Solvent C Conc. |
5 |
|
15.00 |
Pumps |
Solvent D Conc. |
95 |
|
15.01 |
Pumps |
Solvent B Conc. |
0 |
|
15.01 |
Pumps |
Solvent C Conc. |
95 |
|
15.01 |
Pumps |
Solvent D Conc. |
5 |
|
30.00 |
Pumps |
Solvent B Conc. |
0 |
|
30.00 |
Pumps |
Solvent C Conc. |
95 |
|
30.00 |
Pumps |
Solvent D Conc. |
5 |
|
30.01 |
Pumps |
Solvent B Conc. |
95 |
|
30.01 |
Pumps |
Solvent C Conc. |
0 |
|
30.01 |
Pumps |
Solvent D Conc. |
5 |
|
45.00 |
Pumps |
Solvent B Conc. |
95 |
|
45.00 |
Pumps |
Solvent C Conc. |
0 |
|
45.00 |
Pumps |
Solvent D Conc. |
5 |
|
45.01 |
Controller |
Stop |
|
· Store the column with end fitting in respective column box.
· Reverse phase HPLC column washing procedure (Gradient analysis method):
· Select the solvent (Methanol/ Acetonitrile) as per major Constituents of mobile phase.
· Flush the column with 5 % (Methanol/Acetonitrile) and 95 % water for 20 min.
· Flush the column with 95 % (Methanol/Acetonitrile) and 5 % water for 20 min.
· Run the Gradient Program through HPLC column flow rate 1.0 ml/min for 40 Minutes.
Washing method parameter are as described below. C18 HPLC Column Washing Procedure
|
Sr. No. |
System parameter |
Parameter |
value |
|
|
Data Acquisition |
LC Stop time |
40.01 |
|
|
Pump |
Mode |
Low pressure gradient |
|
|
Flow |
1.0000 ml/min |
|
|
Line C Conc. |
5 % |
||
|
Line D Conc. |
95 % |
||
|
Pressure Max |
449 kgf/cm2 |
||
|
|
Auto sampler |
Auto sampler |
Enable |
|
|
Cooler temperature |
25°C |
|
|
|
Oven |
Oven Temperature |
40°C |
|
|
Detector A |
Lamp |
Off |
Gradient Programming is given below.
|
Time |
Module |
Command |
value |
|
0.01 |
Pumps |
Solvent C Conc. |
5 |
|
0.01 |
Pumps |
Solvent D Conc. |
95 |
|
20.00 |
Pumps |
Solvent C Conc. |
5 |
|
20.00 |
Pumps |
Solvent D Conc. |
95 |
|
20.01 |
Pumps |
Solvent C Conc. |
95 |
|
20.01 |
Pumps |
Solvent D Conc. |
5 |
|
40.00 |
Pumps |
Solvent C Conc. |
95 |
|
40.00 |
Pumps |
Solvent D Conc. |
5 |
|
40.01 |
Controller |
Stop |
|
·
Store the column with end fitting in respective column box.· Normal Phase HPLC Column Washing Procedure:
· Flush the Column with HPLC grade IPA. C18 HPLC Column Washing Procedure
· Run the HPLC grade IPA through HPLC column by 0.5ml/min to 1.5-ml/min flow where by pressure will not exceed above 3500 psi.
· Flush the column with HPLC grade IPA for 45 minutes.
· Washing method parameter are as described below.
|
Sr. No. |
System parameter |
Parameter |
value |
|
|
Data Acquisition |
LC Stop time |
45.01 |
|
|
Pump |
Mode |
Low pressure gradient |
|
|
Flow |
1.0000 ml/min |
|
|
Line D Conc. |
100 % |
||
|
Pressure Max |
449 kgf/cm2 |
||
|
|
Auto sampler |
Auto sampler |
Enable |
|
|
Cooler temperature |
25°C |
|
|
|
Oven |
Oven Temperature |
40°C |
|
|
Detector A |
Lamp |
Off |
Gradient Programming is given below.
|
Time |
Module |
Command |
value |
|
0.01 |
Pumps |
Flow |
0.5000 |
|
15.00 |
Pumps |
Flow |
0.5000 |
|
15.01 |
Pumps |
Flow |
1.0000 |
|
30.00 |
Pumps |
Flow |
1.0000 |
|
30.01 |
Pumps |
Flow |
1.5000 |
|
45.00 |
Pumps |
Flow |
1.5000 |
|
45.01 |
Controller |
Stop |
|
Store the column with end fitting in respective column box.
Note: For Normal phase analysis Before Start the Batch file. purge D Line with the IPA for 10 minutes.
Read more about column washing Procedure Here
