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
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 Start-UP Procedure:
4.2.1 Switch ON the Mains, provided on the
backside of the Instrument, starts the Computer.
4.2.2 Click on Start button, select program and
click on Chromeleon or double click on Chromeleon shortcut icon provide on
desktop.
4.2.3 Select and double click on HPLC 02 for Auto
sampler or HPLC 03 for manual HPLC 02 or HPLC 03 system display.
4.2.4 Select and click on Panel, Select and double
click on HPLC 02 or HPLC 03 icon, HPLC system display.
4.2.5 Select control menu and click on command or
Press F8 key to display command menu.
4.2.6 Select and double click on pump icon, Select
and double click on pump left for auto sampler or pump right for
manual.
4.2.7 Select and click on Purge icon, select purge
ON and click EXECUTE button. (For system purging open the purge valve first
provided right side of the pump).
4.2.8 Select pump system setting,
click on Setting button add flow rate of required left pump or right pump with mobile phase concentration.
4.2.9 Select auto sampler, click on ‘Setting’
button auto sampler menu display feed require parameter Injection volume, Prime
Syringe (If click on Prime Syringe, always Pump flow rate is OFF) etc., after
completed this setting click on close button.
4.2.10 Select Column Oven and click on ‘Setting’
button, Column Oven Menu display, set require temperature and click on close
button.
4.2.11
Select
Photodiode Array Detector and click on ‘Setting’ button, Set requires
wavelength on channel 1 and click on
close.
4.3 Preparation
of New Method or Open Method :
4.3.1 Select File Menu and click on New for
preparation of new method. Select method and double click or click OK button,
method menu display.
4.3.2 For Opening method file select file menu and
click on Open, select HPLC system and double click and select require method
file and click.
4.3.3 Select General, General menu display. Select
detector and feed required parameter with ▼ key and Enter key.
4.3.3 Select
Peak
4.3.4 Select Amount Table, Amount Table menu
display. Feed required parameter.
4.3.5 Select
Peak
4.3.6 After completion of all parameter select
save command in file menu, give file name and Select
Path.
4.4 Preparation of New Program or Open
Program:
4.4.1 Select File Menu and click on New for
preparation of new program. Select program and double click program menu
display.
4.4.2 For Opening program file select file menu
and click on Open, select HPLC system and double click and select require
program file and click, program menu display.
4.4.3 Select and Click on my computer, select
require HPLC system and click ‘Next’ button,
display Program Wizard column oven option, feed require temperature.
4.4.4 After adding temperature click ‘Next’
button, displays Sample Option, feed require parameter and click ‘Next’
button.
4.4.5 Obtained Pump Left Option, Select and feed
require parameter and then click ‘Next’ button, display Pump right Option. (If
working on HPLC system 02-auto sampler feed only Left Pump parameter and if
working on HPLC system 03 Manual feed only Right Pump parameter.)
4.4.6 After pump setting click ‘Next’ button,
display sampler option, select Pump, Detector
4.4.7 Display Acquisition Option, select required
parameter and click on ‘Next’ button, display Column Oven Temperature Option,
select Auto and click on ‘Next’ button.
4.4.8 Display Pressure Option, select Auto and
click on ‘Next’ button.
4.4.9 Display UV Option, select requires Wavelength,
Runtime. If require PDA detector always select 3DFIELD and click ‘Next’ button.
4.4.10 Add file name and then click on finish
button.
4.5 Preparation
of New Sequence or Open Sequence:
4.5.1 Select File menu and click on New for
preparation of new Sequence. Select Sequence (Using wizard) and double click or
click OK button, Sequence wizard display.
4.5.2 For
Opening Sequence file select file menu and click on Open, select HPLC system
and double click and select require sequence file and click, Sequence wizard
display.
4.5.3 In
sequence wizard, click next button. Select my computer and double click choose
HPLC 02 or HPLC 03 and click on ‘Next’ button.
4.5.4 Step 2 of 5 sample display,
feed require parameter or and click on ‘Next’ button.
4.5.5 Step 3 of 5 sample display,
feed require parameter or and click on ‘Next’ button.
4.5.6 Step 4 of 5 sample display, method and
reporting display, select method file and Program file with browse button.
Select preferred report format with browse button and click ‘Next’ button.
4.5.6 Step 5 of 5 sample display, saving
sequence, give Sequence name, Title name and give path on HPLC 02 or HPLC 03
with browse button and then click on finish button.
4.5.7 For injection starting open
sequence file, select Batch Menu and click start to start the sequence and run.
4.6 Print:
4.6.1 For Opening Sequence file select file
menu and click on Open, select HPLC system and double click and select require
sequence file and click, Sequence wizard display.
4.6.2 Select require data and double click
chromatogram display. If require any parameter to be changed select view menu
and then click on QND editor and change require parameter.
4.6.3 Select view menu and click printer layout,
print preview display and then select print in file menu and click.
4.7 COLUMN FLUSHING :
4.7.1 After completion of analysis clean the
column by the following solvents as mobile phase.
4.7.2 For reverse phase columns:
4.7.2.1 Flush with the same mobile phase, this was
need for analysis for 15 minutes with 1.0 ml flow rate.
4.7.2.2 Flush with water for 30 minutes.
4.7.2.3 Then flush with Methanol for 15 minutes at a
flow rate of 1.0 ml / minute.
4.7.3 For normal phase columns:
4.7.3.1 Flush with the same mobile phase which was
need for the analysis for 15 minutes at a flow rate of 1.0 ml / minute.
4.7.3.2 Then flush with n -Hexane at a
flow rate of 1.0 ml / minute.
4.7.4 Change over from Reverse phase to Normal phase:
4.7.4.1 After the analysis is over flush the column
with water for 30 minutes, followed by
Methanol for 15 minutes.
4.7.4.2 Remove the reverse phase column (C18 or C8) and attach a dead
volume instead of column.
4.7.4.3 Now flush the system with Acetonitrile
followed by Chloroform and n -hexane (all 15 minutes each).
4.7.4.4 Remove dead volume and fix a normal phase column (e.g.
silica)
4.7.4.5 Continue flushing for 15 minutes.
4.7.4.6 Maintain the reverse phase column in Methanol.
4.7.5 Change over from normal to reverse
phase:
4.7.5.1 After the analysis is over flush the columns
with n- hexane for 15 minutes.
4.7.5.2 Remove the normal phase column (C18 or C8)
and attach a dead volume instead of column.
4.7.5.3 Flush the system with Chloroform followed by
Acetonitrile and Methanol for 15 minutes.
4.7.5.4 Now fix the reverse phase column and flush
the column for 15 minutes.
4.7.5.5 Continue the analysis in reverse phase.
Store the normal phase column in n -hexane.
4.8 Enter the details in HPLC usage logbook as per given in Annexure –1.
5.0 Safety precaution:
5.1 After
analysis run the system wash 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. Then Methanol and
finally with water.
6.3
Sonicate the suction filter with 2 M Nitric acid and then with water.
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
|
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Date
|
Name of Sample
|
Booking No.
(A. R. No.)
|
Batch No.
|
Analyzed by
|
Remarks
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