1.0
Objective:
To
provide written procedure for operation of High Performance Liquid Chromatography system
EQU-INS-201.
2.0
Scope:
This SOP covers operation of
High Performance Liquid Chromatography system make: Shimadzu (Model: LC 2010CHT).
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 record 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.
4.3 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
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 %, adds maximum
pressure and minimum pressure value.
4.5
Create a New Method File or
Modify Method:
4.6.1 Choose Open or ‘New’ option from the method
in File menu in the instrument window, Method dialog box display on the screen.
4.6.2 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, then click on ‘OK’ button.
4.6.3 Select and click on Integration Events in
method menu, Integration Events dialog box display, feed the require
parameters, and then click on ‘OK’ button.
4.6.4 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’.
4.6.5 Select and click on Advanced, Advanced
method option dialog box display, Select Component name, Retention time, Area,
Asymmetry, and Resolution etc.
4.6.6
Select and click on Instrument Setup in
method menu, Instrument dialog box display, and click on ‘PUMP’ button.
4.6.7
Select the mode of pump as Isocratic flow or
low pressure Gradient, feed the flow rate and concentration of solvent in %,
adds 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.
4.8.3 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 a flow rate of 1.0 ml / minute.
4.10.3.2 Then flush with n -Hexane at a flow rate of 1.0 ml / 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
4.10.4.3
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.
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