1.0 Objective:
To
provide written procedure for operation of High Performance Liquid
Chromatography system.
2.0
Scope:
This SOP covers operation of High Performance Liquid Chromatography
system Merck (Model: L7).
3.0
Responsibility:
Jr.
Research Officer, Research Officer: Responsible for operation, calibration and
maintenance of the instrument as per procedure.
Head of Department: Responsible for calibration and maintenance, timely
as per schedule.
QA Officer/QA Manager: Review the records and governing the document.
4.0 Procedure:
4.1 Ensure
that the instrument is visibly clean and free from dust.
4.2 Switch the pump
power on. Insert the end of the solvent tube into the solvent bottle and open
the drain valve.
4.3
Start the pump with “PUMP ON/OFF” key and press the “PURGE” key. Allow
the pump to run until no more air bubbles exit from the outlet. Stop the
purging by pressing “PURGE” key again.
4.4 Setting the Flow-Rate:
4.4.1
Press ‘MANUAL SET’ key the cursor show on the display. Press ‘ENTER’ key
to select flow and enter the required flow-rate with numerical key and press
‘ENTER’ key to confirm the flow-rate.
4.5
Setting gradient system:
4.5.1
Press ‘INT’ key and ‘SET PROG’ key display shown gradient system program
1 to 9,
Select the program and press ‘ESCAPE’ key returns to the monitor screen
without changing the setting.
4.5.2 Select the program, if program
modify, Select ‘0’ by pressing ‘0’ numerical key or prepare new program, Select
‘1’ by pressing ‘1’ numerical key. Press ‘ENTER’ key to select the program.
4.5.2
Press ‘ENTER’ key, select and add time interval, pump A flow-rate
in %, pump B flow rate in %, pump C flow rate in %, pump D flow rate in % with numerical key and press ‘ENTER’ to set
value.
4.5.3 Set the time program,
measurement wavelength with numerical key and ‘ENTER’ key.
4.5.4 Correct
the input numeric by using ‘CL’ key. Use the ‘DELETE’ key to change the
memorized setting numeric.
4.5.5
Use the ▲ and ▼ key to be
checked program or step of setting program.
4.5.6 Load the injection on the
injector, immediate press ‘START/STOP’ key to start the gradient system pump.
If stop the gradient system presses ‘START/STOP’ key to stop the gradient
system pump.
4.6
If required column temperature attach column oven and set required
temperature.
4.7
UV DETECTOR:
4.7.1 Switch on the detector using
the POWER switch. After the self-test has passed, a status-indicating screen
appears on the display.
4.7.2 Pressing the ‘AUTO ZERO’ key, the absorption at the chosen wavelength is
set to zero.
4.7.3
Setting Wave length:
Set the wavelength range with pressing ‘WAVE LENGTH’ key, enter the
require wavelength with numerical key and press ‘ENTER’ key. The wavelength has
been set and is indicated on the display.
4.8
Setting up the Time Program:
4.8.1 Press
‘TIME PROG’ key to set a time and wavelength for automatic change of
measurement wavelength during analysis.
4.8.2 Input the program no. to be
created or changed. Settable program nos. are from 1 to 9. Pressing the
‘ESCAPE’ key returns to the monitor screen without changing the settings.
4.8.3 Select the program, if program
modify, Select ‘0’ by pressing ‘0’ numerical key or prepare new program, Select ‘1’ by pressing ‘1’ numerical key. Press
‘ENTER’ key to select the program.
4.8.4 Set the time program,
measurement wavelength with numerical key and ‘ENTER’ key.
4.8.5
Correct the input numeric by
using ‘CL’ key. Use the ‘DELETE’ key to change the memorized setting numeric.
4.8.6
Use the ▲ and ▼ key to be checked program or step of setting
program.
4.8.7
To execute the time program set with the ‘TIME PROG’. Press the
‘START/STOP’ key, a display is provided on the monitor screen and measured
values are output.
4.8.8
Load the injection on the injector, immediate press ‘START/STOP’ key to
start the time program for detector. If stop the time program press
‘START/STOP’ key to stop the time program.
4.9
Check the Lamp Energy:
4.9.1
Press ‘CONFIDENCE’ key,
Confidence menu display on the screen. Select ‘CHECK’ menu with pressing
numerical ‘2’ key. Display lamp energy and wavelength accuracy on the display.
4.10
RI DETECTOR :
4.10.1
Switch on power switch provided
on right side corner of the detector. After the self-test has passed, a status-indicating
screen appears on the display.
4.10.2
Press ‘SENS’ key to check the
sensitivity of detector or add require sensitivity with pressing ‘SENS’ key and
then press ‘ENTER’ key to confirm the value.
4.10.3
Press ‘FUNC’ key and then press
‘AUTOZERO’ key to detector display will be zero.
4.10.4 For purging press ‘FUNC’ key
and then press ‘AUTOZERO’ key
4.11
FLUORESCENCE DETECTOR :
4.11.1
Switch on power switch provided
on left side corner of the detector. After completion of initialization screen
appears on the display for wave length setup.
4.11.2
Set the extinction wavelength
and emission wavelength by pressing wavelength key on the front panel of the
detector.
5.0 SOFTWARE OPERATION:
5.1
Start windows on your PC and in the
program group ‘Winchrom’, click the winchrom icon, Winchrom main screen is
display.
5.2 To Create a New
Method File:
5.2.1 Single
click the ‘METHOD’ icon in winchrom main screen. Method dialog box has display
on the screen.
5.2.2
Choose Open or ‘New’ option from the File menu. Display show new Method
dialog box on the screen.
5.2.3 Add Runtime and Minimum peak width value with ▲ and ▼.
5.2.4 Select HPLC and click
on details, display show HPLC parameter dialog box.
5.2.4.1 Select the temperature
for Oven temperature.
5.2.4.2 Enter the value for
mobile phase.
5.2.4.3 In the mobile Phase
enter the solvent name for A, B, C, D phases.
5.2.4.4 In the column field,
enter Description, Guard column, I.D. ,
etc.
5.2.4.5 In
the injection field, enter the loop Size in µl. Select the mode of operation
manual or Auto.
5.2.4.6
Click on “Program” enter the
flow rate.
5.2.4.7
Choose OK to done or choose
cancel to abort the dialog box and return to previous
screen.
5.2.5
Select and click Parameters,
Method Parameter dialog box display.
Click on detector, detector parameter dialog box display, feed the
parameters, select detector, and then click on OK.
5.2.5.1
Click Integration, Integration
parameter dialog box display, feed the parameters then click on OK.
5.2.5.2
Click Screen, Screen parameter dialog box
display, feed the parameters then click on OK.
5.2.5.3
Click Report, Report parameter dialog box display, feed the parameter
then click on OK.
5.2.5.4 Click System Suitability, System
suitability parameter dialog box display, feed the
5.2.5.5 Click ‘OK’ to confirm all method
parameters.
5.2.6
Select and click Time Events from
Method dialog box, Time Events dialog box display, feed the require parameters
then click on OK.
5.2.7
Select and click Component Table from Method dialog box, Component Table
dialog box display, feed the retention time, component name and then click
‘OK’.
5.3 After adding all parameter in
Method parameter, save method from file menu. Enter or Select path and full
file name.
5.4 After completion of this
procedure inject the solution and injector move in inject position.
5.5
If print of graph is require, click the
‘REPROCESS’ icon in Win-chrom main screen, select and Open data file from the
file menu with selecting path. Reprocess with click reprocess in Operation
menu. If any changes are require set the require parameters in data file.
5.6
Select
print command from file menu and click the print
5.7 COLUMN FLUSHING :
5.7.1 After completion of analysis clean the
column by the following solvents as mobile phase.
5.7.2 For
reverse phase columns:
5.7.2.1 Flush with the same mobile phase, which was
need for analysis for 15 minutes with 1.0 ml flow rate.
5.7.2.2 Flush with water for 30 minutes.
5.7.2.3
Then flush with Methanol for 15
minutes at a flow rate of 1.0 ml / minute.
5.7.3 For normal phase columns :
5.7.3.1 Flush with the same mobile phase which was need for the
analysis for 15 minutes at a flow rate
of 1.0 ml / minute.
5.7.3.2 Then flush with n
-Hexane at a flow rate of 1.0 ml / minute.
5.7.4 Change over from Reverse phase to Normal phase :
5.7.4.1 After the analysis is over flush the column
with water for 30 minutes, followed by Methanol for 15 minutes.
5.7.4.2 Remove the reverse phase column (C18 or C8)
and attach a dead volume instead of column.
5.7.4.3 Now flush the system with Acetonitrile
followed by Chloroform and n -Hexane (all 15 minutes each).
5.7.4.4 Remove
dead volume and fix a normal phase column (e.g. silica)
5.7.4.5 Continue flushing for 15 minutes.
5.7.4.6 Maintain
the reverse phase column in Methanol.
5.7.5 Change over from normal to reverse
phase :
5.7.5.1 After the analysis is over flush the columns
with n- Hexane for 15 minutes.
5.7.5.2 Remove the normal phase column (C18 or C8)
and attach a dead volume instead of column.
5.7.5.3 Flush the system with Chloroform followed by Acetonitrile and Methanol
for 15 minutes.
5.7.5.4 Now fix the reverse phase column and flush
the column for 15 minutes
5.7.5.5 Continue the analysis in reverse phase.
Store the normal phase column in n -Hexane.
5.8 Enter the details in HPLC usage
logbook as per given in Annexure –1.
5.9 Safety precaution:
5.9.1
After analysis run the system washes with
water for 30 minutes if buffer solution used in
mobile phase followed by Methanol
for 15 minutes.
6.0
Routine maintenance:
6.1 Clean
the instrument by dry cloth.
6.2 Clean the system with hot water without
connecting the column. Than Methanol and
finally with water.
6.3
Sonicate the suction filter with 2 M Nitric
acid.
7.0 Documentation:
7.1 Annexure – 1 HPLC Usage logbook XXX/FRM/000
8.0 History of Revision:
Revision No.
|
Effective
Date
|
Revision
details
|
Reason for
revision
|
Annexure - 1
|
|||||
Date
|
Name of Sample
|
Booking
No.
(A. R. No.)
|
Batch No.
|
Analysed by
|
Remarks
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