HPLC - Operation & Calibration

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
SOP FOR OPERATION

1.0. PURPOSE:

To provide procedure for Operation of High Pressure Liquid Chromatography.

2.0. SCOPE:

Applicable to HPLC (CodeNo. ) :
Make : Shimadzu
Model : LC-2010A
Pump : LC-10Atvp Detector : SPD-10Avp
System Controller : SCL-10Avp
Auto Injector : SIL-10Advp

3.0. RESPONSIBILITY:
3.1. QC- officer
3.2. QC- Executive

4.0. ACCOUNTABILITY:
Manager-Quality Control

5.0. DEFINITION:
High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster.

6.0. PROCEDURE:





6.1.1. Check and ensure that the instrument is clean and calibration is valid.
6.1.2. Switch on SCL10Avp system control,SPD 10Avp UV-Visible detector, followed bySQL/10ADvp autoinjector & followed by LC10ATvp
         pump.
6.1.3. Then start the computer (Softwere),wait for a while;Till enter Password, appear on the monitor.
6.1.4. Enter the password -On screen WELCOMES TO WINDOWS appear.
6.1.5. Enter Esc wait for a while.
6.1.6. Icons appear. Then double click on class-vp system & wait for a while.
6.1.7. Class -vp system will open.
6.1.8. Choose Auto Sampler with double click.
6.1.9. On monitor login appear,enter the users name & Password . Enter User name as System , Password as 2001
6.1.10. Then Icons appear on the screen click on File,choose for METHOD, open a New file or stored file & same by going back to method.
6.1.11. Once again go back to file, Choose the SEQUNCE & save,enter file name data name & save.
6.1.12. Next go to METHOD icon on the screen click over method.
6.1.13. Choose INSTRUMENT SETUP then PUMPS/ SPD 10 AVP (Detector-A) will appear.
6.1.14. Set the Pump for flow rate & max pressure.
6.1.15. In SPD10VP (Detector-A),choose for wavelength & click APPLY followed by DOWNLOAD & save the method.
6.1.16. Once again come back to main screen,then choose icon on the main screen.
          CONTROL,run down click over DIRECT CONTROL the series of icons appear on the screen.
6.1.17 Click icon on instrument ON/OFF Now the system is ready for operation Once analysis is over close the system as follows.
6.1.18. Click over file run down to EXIT.
6.1.19. Click once again on screen SHUTDOWN the computer,it's now safe to turn off your computer message appear.
6.1.20. Similarly switch off SCL10AVP system controller, followed by SPD10AVP, UV-VISIBLE DETECTOR followed by Auto-Injector, & then
           LC10ATVP pump.

Acceptance criteria : maximum pressure variation must not exceed 5.0 Kgf/cm2.

6.1.21. SELECTION OF COLOUMN: Check for the coloumn specified in the individual Monograph, and wash properly with solvents specified to get the proper resolution.

6.1.22 SYSTEM SUITABILITY: Carry out the system suitability test mentioned in individual Monograph.

6.1.23 ‘Frequency of washing’ : Column washing should be carried out after each analysis .
6.1.24 Extra washing should be carried ,whenever abnormal pressure variation or distortion of peaks occurs during analysis.

7.0. REFERENCES:
Instrument Manual

8.0. ENCLOSURES:
HPLC usage log
No.SOP:QCOP:00x6:xx

9.0. ABBREVIATIONS:
Nil


HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

SOP FOR CALIBRATION

1.0.PURPOSE:
To provide procedure for Calibration of High Pressure Liquid Chromatography.

2.0. SCOPE:
Applicable to HPLC
Make : Shimadzu
Model : LC-2010AHT
Pump : LC-10Atvp Detector : SPD-10Avp
System Controller : SCL-10Avp
Auto Injector : SIL-10Advp

3.0. RESPONSIBILITY:
3.1. Officer -QC
3.2. Executive-QC

4.0. ACCOUNTABILITY:
Manager-Quality Control

5.0. DEFINITION:
High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster.

6.0. PROCEDURE:

6.1. Start the HPLC pump as per IOP and check the flow rate by collecting water in a 10ml measuring cylinder. Using a stop watch set the time for 5 minutes and measure the volume collected for 1.0ml/ minute 1.5ml/minute and 2.0ml/minute flow rates respectively. Note the readings in duplicates.

I
Time in minute 5min
Flow rate 1ml/min
Volume of water collected in ml Pump A: ____ Pump B: _____
Limit in ml 4.95-5.05

II
Time in minute 5min
Flow rate 1.5ml/min
Volume of water collected in ml Pump A: ____ Pump B: _____
Limit in ml 7.45-7.55

III
Time in minute 5min
Flow rate 2ml/min
Volume of water collected in ml Pump A: ____ Pump B: _____
Limit in ml 9.95-10.056.2.

CALIBRATION OF COLUMN

Chromatographic system:

Column : C18 Or C 8 (25cmx 4.0mm), (5micron)
Column No. to be mentioned in reports.
Detector : 254nm
Flow rate : 1ml/min
Mobile phase : Degassed and filtered solution containing
Acetonitrile : Water ( 70 : 30 )
Injection volume : 20 ml.

Standard preparation : Transfer 1ml of Toluene and 1ml of Benzene into a 100ml volumetric flask and make upto volume with methanol, shake well, further dilute 5ml to 50ml with methanol. Inject 20ml of standard solution for five times.
Record the following

1. RT of Toluene and Benzene : RSD NMT 2.0%
2. Area of Toluene and Benzene : RSD NMT 2.0%
3. Theoretical plates of Toluene and Benzene : NLT 1500
4. Resolution between Toluene and Benzene : NLT 2.0
6.3. CALIBRATION OF DETECTOR/INJECTOR
To a clean, dry 100ml volumetric flask, add 0.1ml of Benzene and 0.1ml of Toluene. Mix by adding mobile phase and make up to volume. Inject equal quantities three times and record the ratio of the peak area.
Chromatographic conditions
Detector : 254nm
Flow Rate : 1.0 ml/minute
Mobile Phase : Methanol : Water = 80 : 20
Absorbance Scale : 0.02
Attenuation : 5
Injection Volume : 20ml
Column : C-18 (Shim-pak)
Calculation
Area of first peak
Area of second peak
6.4.
Injection No. Ratio % deviation from the average (limit±2%)
1
2
3
Dedicated columns are used as mentioned in the monograph.

6.4. FREQUENCY OF CALIBRATION: Once in three months

7.0. REFERENCES:
Instrument Manual

8.0. ENCLOSURES:
Nil
9.0. ABBREVIATIONS:
Nil