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TITLE:
OPERATION OF AGILENT HPLC
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SOP NO.: |
QC00X-01 |
Effective Date: |
DD/MM/YY |
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Supersedes : |
QC00X-00 |
Page No.: |
1 of end |
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Department: |
Quality
Control |
Review Date: |
DD/MM/YY |
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I OBJECTIVE
To provide a procedure for the operation of Agilent HPLC (High-Performance Liquid Chromatography).
II SCOPE
This procedure is applicable for the HPLC series, used in the quality control department of ABC Ltd.
III RESPONSIBILITY
It is the responsibility of all the
QC personnel involved in the analysis using the system.
IV DOCUMENT REFERENCE(s)
SOPs : Operation and calibration of Analytical balance (Current version of QC00X)
Forms :
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S.No |
Details |
Format No. (Current
version) |
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1 |
HPLC Instrument Log |
QC00X/F01 |
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2 |
HPLC Column usage Log |
QC00X/F02 |
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3 |
Mobile Phase log |
QC00X/F03 |
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4 |
Mobile Phase |
QC00X/F04 |
V PROCEDURE
1.0 For Agilent Model
Power
on all the individual modules of HPLC sequentially i.e.-degassing module, Pump,
Auto-injector, Column thermostat and
detector.
Switch on the computer.
After switching on the instrument of all modules, the VWD
initialization process takes Place.
New project creation:
Then go to the computer, click Start, and then EZ Chrome Elite shall display from this
Only Administrator can
create new project and assign a user to access that project with different rights
in below screen, all other options are disabled, after the administrator login, it
will be enabled. To access the menu, click on Tools. Enterprises login
Enter the administrator username and password
Click on the Tool System
administration wizard….
Select project. Click on
Next
Select create a new
project. Click on Next
Enter the name of the
project. Click on Next.
If you want to enable
Audit trail settings then select automatically enable audit trail. Also, you can
give a list of reasons here. Click on Next
In the below screen you can
add E-Signature roles. Click on Next
In below screen, list of
all users will be displayed in Available users. You have to select the users
from that list and move it right side, so only those users will access the project
which you are creating now. Click on next
Here you can give
different rights to access the project to different users. For that you have to
select the user first from list of users and then select privileges from unassigned
privileges and move it to right side. Screen after giving
privileges to the user. Click on Next
Click on Finish
Same way you can modify
the project setting, assigned users to projects etc. With admin login. After
completion of this admin activity, you need to admin logout. For that click on
To
Login in to EZChrom elite:
Double click on EZChrom elite icon on the desktop OR
You can click on start programs chromatography EZChrom Elite
EZchrom Elite window will
appear from there you can open it online and offline.
If you want to open an online
session then double click on the HPLC-01(LC or GC name) icon.
One login window will appear,
in which you have to enter your user name, password and you have to select your
working project from scroll down menu. Then click on login tab.
If you want to open your
offline session then click on HPLC-01 (LC or GC name) then clicks on open
offline. Then same login window will appear. click on OK
In this window you will
find navigation pane left side. From that directly access the method, sequence,
control, report menus.
New
method creation:
Click on file >method > New
The instrument set up window
will appear aslow or you can go to this instrument set-window by clicking
MeaInstrument setup or from
the navigation pane.
Instrument setup window you
will find all the modules which are configured with software. Here you can see
VWD (UV detector), TCC (column oven), Quat pump, ALS (auto sampler with sample
cooler if installed)
In the VWD tab: Enter the wavelength of analysis and select stop time AS pump/injector. To further optimize
the method change the peak width, otherwise no need to change another parameter in the same window if clicked on. Advanced then you will find some other parameters
related to VWD. Refer above screenshot.
In TCC tab: Enter
the temperature of the column, if you can give temp for left side, right side
or both side here. If you select Not controlled then TCC will work at ambient temperature.
If you select combined then TCC left and right side temp will be same as given
in left side. Select stop time as pump/injector. No need to change other
parameter. To access other parameter related to TCC you can click on Advanced.
In Quat pump tab: Enter Flow in ml/min, solvent concentration
by selecting A, B, C, D lines pressure limits (Max). No need to change Min
Pressure limit (always set zero). Here you can type you mobile phase detail in
blank space. Select stop time i.e. analysis run time in minutes. From right
side you can create gradient program by add, remove etc. button. No need to change
other parameter. Also you can access other parameter related to pump by
selecting Advanced.
In the ALS Tab: Enter Injection volume in uL.
Select either Standard injection or injection with needle
wash.
In standard injection, the Needle will not be cleaned during
injections.
In injection with needle
wash mode when you select
this mode location option wil be enabled. Here you have to enter the wash vial
number, Needle outer surface will be cleaned after sample sucked from the
sample vial.
Enter stop
time in minutes same as given in Quat pump parameter.
If ALS thermostat
(sample cooler) is installed then enter temperature (sample).
No need to change other parameter. For further method optimization you can have some other Advanced parameter related to Auto sampler in same window.
In Aux Traces tab: You can also able to acquire the below signals during the analysis like pressure, graph, column oven temp graph etc. select which ever you want to see along with detector signal.
In last tab trigger: Normally where auto sample is installed, it is External. No need to change. Detail also mentioned in same window for different option.
After
editing all the method parameters, you need to save all the method by
clicking
Save method screen will appear.
Give method filename in blank space (For example: Demonstration)
After saving method file,
download it into instrument by clicking control Download method.
After downloading the method you can
see the online signal before injection i.e. to check baseline gets stabilized
or not. Click on view online signal.
How to do single run Acquisition:
Click on control single Run. One window will appear in that
need to enter sample ID, vial number and injection volume.
How to create new sequence:
Click on the file Sequence Sequence wizard
Sequence wizard window
will appear as below. In this window, Select method using browse button, select
data file type for acquisition click on Next.
Enter sample ID, Data path
(browse your path if required), Data file name. In sample ID and Data file name
by clicking triangle symbol ►you will be able to give different of naming
sample ID and data file here let select increment number. Give number of
unknown runs in sequence (i.e. numbers of lines want to create in sequence, for
exp.give:10). Also you can give repetitions per run. After filling detail in
below window, click on A newt.
New window will appear as
below.
In Unknown vials of sequence: Enter First vial and incrthe the ement by and give injection volume in μl by giving increment By: 1, 10 lines will be generated in sequence with file names automatic using viaautomaticallyally. Click on new
New window will appear as
below. No need to change in this window. Click on nexa a t
Now window will appear as below. No need to change in this window. Click on fiThe sequencequence will be generated as below.
You can choothe the se number of
columns the the in sequence table clickingick on sequence table
PropertiThe belowlow screen will appear.
Selethe ct required columns from this list.
Edit sequence table, i.e. give correct
vial no, repetitions per runs, inrunion volume, method nand and ame if required.
Verify the method path, data patand and h by clicking sequence es.
To save this sequence click on File
Give sequence file name in Blank space
How to start sequence run
Click on control sequence Run OR Right click
on sequence tabl Run sequence.
One Run sequence window
will appear, here you can select all the lines or some selected lines or range
of lines from the sequence table. DefaultThe defaultion will be all. Click on start.
Your sequence will be startou can see the status in sequence the table when analysis
is going on.
How to access acquired data, integrated data, Assign peak
name, process sequence
From main EZ Chrom Elite
window right click on HPLC-01(LC or GC name) Open offline.
One login window will appear,
in which you have to enter your user and name, password and you have to select your
working projectthe the from scroll down menu. Then clithe the ck on login tab. click on file DataOpen
Select data file and from option select Original/Acquisition
That will open data file
with original method with which data acquired
Data file will
open with related method.
To integrate
chromatograms, integration events are available
By clicking on the chromatogram Graphical
programming list will be displayed, you can use different integration events like integration off, valley
to valley etc.
OR you can integrate
chromatogram using graphical tools i.e. in same window.
All the integration entries
you have applied that you can see from method Integration Events.
Below screen will appear.
From this table also integration can be set. You can add integration in event
column using scroll down menu and manually entering start/ end time, value.
Now at this time
chromatogram window will disappear behind the table. To see the chromatogram
click on view Tile data.
To check the effect of integration you have applied on chromatogram, click on A Analyze
For Assigning Peak Name:
Right click on
chromatogram Graphical programming Define peaks
Enter the range of peaks you
want to define (i.e. starting point and end point). After this click OK.
Click on method Peaks/ Groups.
The peaks/ groups window will
appear as below.
Give correct peak names
from their RT in this table in the name column. You can change also the RT window from
this table.To see the namess of peaks
on the chromatogram, click on Analysis Analyze.
Click view Tile data.
Then right-click on
chromatogram Annotations. The below window will appear
Select Name, Area, RT, or whatever you want to see on the chromatogram. Select it and move to the right side. Then click on OK.
After proper integration and assigning
peak names, you have to save this integration into the method so click on File,
Method, and Save. It will save the method which you opened last, you can see on
top method name.
To apply the same integration
set-in method to all data files in sequence
Open the sequence by clicking
File Sequence. Select the sequence you want to process.
Here take care that your method in the sequence must be set with proper
integration events before.
Click on sequence process OR click on Sequence Process sequence.
Process sequence window will appear as below. You can select All/Made selection/Give range. Click on Start.All data files in sequence table will be processed using integration events saved in respective method
Print out of chromatograms:
Open the data file by clicking File Data open. Then Select your interested data
file. Open the respective method for that data.
Click on Reports View Area% (you can select any report
template listed)
Below is area % report
generated.
To give print of this report simply
right-click on report Print.
You can edit OR create new report
template by clicking Reports Report
Template Editor
After above action, you can edit the
report template by right click on report
List will be displayed. Then you can import reports all ready existing
and edit it or create new report template.
After editing report template save
the report template by clicking File Report template Save As
How to find system suitability of repeat runsOpen the sequence you have already run before. Set proper integration for method used in sequence For example: open sequence injection precision seq.In this sequence table demonstration met method is used. This method is already set with proper integration events. Also it is compulsory to assign peak names in chromatograms and save it in method. Open that method.After proper integration and peak assigning chromatogram s.To take print out of this curve and calculation. Right click on curve .Print all peaks/Groups
CARE AND MAINTENANCE:
Ensure the details are documented in the column usage log after the analysis & washing activity is completed.
Use 85:15 Acetonitrile and water for the plunger wash.
Use mobile phase / diluent vial for syringe needle wash
Verify the performance of the system as per the schedule / after a major breakdown of the instrument.
Replace the inlet filters based on their performance
Enter the breakdown details & spares utilized in the Instrument malfunction record.
REFERENCE:
* Instrument Manual of Agilent HPLC SystemREVISION HISTORY
END O F THE DOCUMENT
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