HPLC Operational SOP

1.0  OBJECTIVE:

To lay down the operating procedure of High Performance Liquid Chromatography (HPLC).

2.0  SCOPE:

This shall be applicable to the High Performance Liquid Chromatography (HPLC) of Quality Control Instrument Laboratory.

3.0  RESPONSIBILITY:

3.1  Technical Assistant/ Helper.

3.2  Q.C Analyst.

4.0  ACCOUNTABILITY:

4.1  Manager-Quality Control.

5.0  MATERIAL AND EQUIPMENT:

5.1  High Performance Liquid Chromatography (HPLC).

5.2  Sample.

6.0  PROCEDURE:

6.1  Operation:

6.1.1        Power ON the system:

6.1.1.1  Power on the pump, detector, and any other component like computer.

6.1.2        Purging the pump:

6.1.2.1  Ensure that the solvent inlet filter, inside the solvent bottle, is in vertical position. Pump must be purged with every new solvent.

6.1.3        Set the analytical parameters:

6.1.3.1  After power ON the status menu will appear to change the parameters.

6.1.3.2  Change flow rate and wave length according to requirement.

6.1.3.3  Run purified water approximately 20 minutes.

6.1.3.4  After that run mobile phase for approximately 30 minutes to saturate column with mobile phase.

6.1.3.5  Inject standard or sample only when reading on detector is zero.

6.1.3.6  Press auto zero before each injection.

6.1.4        Shut down:

6.1.4.1  After completion of work following steps must be taken to save the life of system.

6.1.4.2  Wash with 80ml of water (HPLC Grade) as under.

6.1.4.3  Dip inlet tube in the water container and press [RUN]. The water will go through the pump, Injector, column, and detector. In this way all salts and inorganic materials will be washed. After 70 ml of water passing press [STOP] key.

6.1.4.4  Finally pass 50 ml (approximately) methanol through the column to save the column. After 50 ml of methanol passing press [STOP] key.

6.1.4.5  Power OFF the pump, Detector.

6.2  Precautions:

6.2.1        Check all tubing, wiring and column that should not be blocked broken or pricked.

6.2.2        Column must be compatible to the analytical method as well as mobile phase. Make corrections if necessary.

6.2.3        The availability of reference samples, solvents and chemicals must be confirmed.

6.2.4        Table having instrument should be free of vibration.

7.0  REVISION LOG:

Revision No.	Effective Date	Reason
00		New SOP

8.0  REFERENCES:

8.1  Not Applicable.

9.0  ANNEXURES:

9.1  Not Applicable.

10.0    ABBREVIATIONS:

Abbreviation	Expanded Form
SOP	Standard operating procedure
&	And
No.	Number
Ltd.	Limited
Q.C	Quality control
QCEO	Quality control equipment operation
F	Format
ml	Milliliter
HPLC	High Performance Liquid Chromatography