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
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 Starting
UP and Connecting Instruments and Class –VP Software:
4.2.1 Turn on the instruments.
4.2.2 After confirming that the instrument
is started up, turn the PC power ON, and select and click on Start icon, Select
programs and chromatography and click on ‘CLASS VP’. OR double click on ‘CLASS
VP’ icon on desktop. Display shows Class VP dialog box.
4.2.3 Select and double click on the instrument
icon in the Class VP dialog box.
4.2.4 Enter
a user name ‘System’ and password ‘2001’; to log in to ‘CLASS-VP’, the
instrument window opens.
4.2.5 Click on pump icon or and fed the require
flow rate and concentration of solvent.
4.4 Purging
Mobile phase and Rinse Solution :
4.4.1 Click
on the ‘PURGE’ icon on the control toolbar. Select the flow lines to be purged
and set purge time for each.
4.4.2 Clicking
on the purge button display a window showing the progress of purge, and the
auto purge starts.
4.4.3 If stop the purging click
‘Mobile Phase Stop’ button.
4.4.4 Click on ‘PUMP’ icon into
instrument window, Select the mode of pump as Isocratic flow or low
pressure Gradient, feed the flow rate and concentration of solvent in %,
add maximum
pressure and minimum pressure value.
4.5 Create a New Method File or
Modify Method :
4.6 Choose Open or ‘New’ option from the method
in File menu in the instrument window, Method dialog box display on the screen.
4.6.1.1 Select the commands in the
Method menu from the Option tab, Select and click on Properties, Method
properties dialog box display, feed the require parameters, and then click on
‘OK’ button.
4.6.2 Select and click on Integration Events in
method menu, Integration Events dialog box display, feed the require
parameters, and then click on ‘OK’ button.
4.6.3 Select and click on Peaks/Groups in method
menu, Peaks/Groups dialog box display; add Component name, Retention time etc.
and then click on ‘OK’.
4.6.4 Select and click on Advanced, Advanced
method option dialog box display, Select Component name, Retention time, Area,
Asymmetry, and Resolution etc.
4.6.5 Select and click on Instrument Setup in
method menu, Instrument dialog box display, and click on ‘PUMP’ button.
4.6.6 Select the mode of pump as Isocratic flow or
low pressure Gradient, feed the flow rate and concentration of solvent in %,
add maximum pressure and minimum pressure value.
4.6.8 Click
on ‘Oven’ button, add temperature maximum up to 60° and add Oven temperature.
4.6.9 Click
on ‘Detector’ button, Select D2 lamp, Polarity, Cell temperature low, add wavelength on Channel 1 and require for
Channel 2, add sensitivity and select
Acquisition On, add require Run time.
4.6.10 Click
on ‘Controller’ and select Degasser.
4.6.11 Click on ‘Time Program’ and
select module (Pump, Oven, Detector etc.) add require time and program.
4.6.12 Select and click on System Suitability in
method menu, System Suitability dialog box display, select the require parameters i.e.
Area, Retention time, Asymmetry, Resolution
etc. and then click on ‘OK’.
4.6.13 After completion all the
parameters save method from file menu. Enter or Select path and full file name.
4.7 Create a New Sequence File or Modify
Sequence:
4.7.1 Choose Open or ‘New’ option from the Data in
File menu in the instrument window, Data dialog box display on the screen,
create a new folder and give name of the folder.
4.7.2 Choose Open or ‘New’ option
from the sequence in File menu in the instrument window, sequence dialog box
display on the screen.
4.7.3 In Run information add Sample
ID name, Select method, Select Data path, and give Data file name.
4.7.4 Add Amount values as per requirement.
4.7.5 In Auto sampler feed the parameters, start
vial, end vial, injection volume and Repetitions per run, and then click on
‘OK’, sequence file display on the screen.
4.7.6 In sequence file feed parameter sample
identification, vial number, injection volume respectively.
4.7.7 After completion all the parameters save
sequence from file menu. Enter or Select path and full file name.
4.7.8 After completion all parameters, right click
of the mouse, select and click on start sequence.
4.8
Creating Report Templates:
4.8.1 Click on the Edit Custom Report button or
Select and click on Custom Report, Custom
Report dialog box display.
4.8.2 Right
click of the mouse, select report header and feed parameters, select graph and
drag the graph on custom report dialog
box display, peak report. After completion feed all parameters save template
file.
4.8.2 Select print in file menu, and give print
command for printing.
4.9 Opening of Instrument Off
Line :
4.9.1 Select and double click on the instrument off
line icon in the Class VP dialog box.
4.9.2 Enter a user name ‘System’ and password
‘2001’; to log in to ‘CLASS-VP’, the instrument off line window opens.
4.10 COLUMN
FLUSHING :
4.10.1 After
completion of analysis clean the column by the following solvents as mobile
phase.
4.10.2 For reverse phase columns:
4.10.2.1
Flush with the same mobile
phase, which was need for analysis for 15 minutes with 1.0
ml flow rate.
4.10.2.2
Flush with water for 30 minutes.
4.10.2.3 Then flush with methanol for 15 minutes at a
flow rate of 1.0 ml / minute.
4.10.3 For normal phase columns:
4.10.3.1 Flush with the same mobile phase which was
need for the analysis for 15 minutes at flow rate of 1.0 ml / minute.
4.10.3.2 Then flush with n -Hexane at a flow rate of
1.0ml/minute.
4.10.4 Change
over from Reverse phase to Normal phase:
4.10.4.1 After the analysis is over flush the column
with water for 30 minutes, followed by Methanol for 15 minutes.
4.10.4.2
Remove the reverse phase column
(C18 or C8) and attach a dead volume instead of column.
4.10.4.3 Now flush the system with acetonitrile
followed by chloroform and n -hexane (all 15 minutes each).
4.10.4.4 Remove dead volume and fix a normal phase column (e.g. silica)
4.10.4.5 Continue flushing for 15 minutes.
4.10.4.6 Maintain the reverse phase column in
methanol.
4.10.5 Change over from normal to reverse phase
:
4.10.5.1 After the analysis is over flush the columns
with n- hexane for 15 minutes.
4.10.5.2 Remove the normal phase column (C18 or C8) and
attach a dead volume instead of column.
4.10.5.3 Flush the system with chloroform followed by
acetonitrile and methanol for 15 minutes.
4.10.5.4 Now fix the reverse phase column and flush
the column for 15 minutes.
4.10.5.5
Continue the analysis in
reverse phase. Store the normal phase column in n -hexane.
4.11 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. Than methanol and finally with water.
6.3
Sonicate the suction filter with 2 M
nitric acid and than water and than 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
|
SOP No.:XXX/SOP/000-00 Format No.: XXX/FRM/000 -00
|
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