023 Operation Procedure For High Pressure Liquid Chromatography (HPLC) - 04

SRI VYJAYANTHI LABS PVT. LTD.
PLOT NO. 27/A, JNPC, PARAWADA (M) VISAKHAPATNAM – 531 019.
Doc No.: SVLL/QC/SOP/023-04

Standard Issue date : Effective date :
Operating
Procedure Review date :
Supersedes: SVLL/QC/SOP/023-03
Page 1 of 8
Department: Quality Control
TITLE: OPERATION PROCEDURE FOR HIGH PRESSURE LIQUID
CHROMATOGRAPHY (HPLC)
1.0 Purpose:
To describe the procedure for Operation & Maintenance of a High Pressure Liquid
Chromatography.
2.0 Scope:
This SOP is applicable to 1260 Infinity Agilent make High Pressure Liquid Chromatography
along with Auto Liquid Sampler, Column Compartment, Variable Wavelength Detector and
Refractive Index Detector controlled by the Open Lab Soft ware.
3.0 Definitions:
HPLC : High Pressure Liquid Chromatograph
LC : Liquid Chromatograph
HPLC and LC are used as synonyms for the purpose of this procedure.
4.0 Responsibility:
It is the responsibility of HOD – QC / QC Chemist to implement this procedure.
Prepared by Reviewed by Approved by

Name R.RAMA DEVI K.V. SUBBA RAJU V.MADHAVA RAO
Designation EXECUTIVE - QC HOD - QC MANAGER - QA
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Doc No.: SVLL/QC/SOP/023-04 Page 2 of 8

5.0 Procedure:
5.1. System Description & Principle:
5.1.1. The Liquid Chromatography consists of a quaternary pump, mobile phase reservoir,
an auto injector, a chromatographic column compartment, and Variable Ultraviolet
Wave Length detector, Refractive Index Detector data acquisition system (P.C.).
5.1.2. The mobile phases are pumped at a selected flow rates and passes through the
injector, column and selected detector.
5.1.3. Separation of the different components of the sample is achieved through-out the
column, because of the following reasons.
a) Molecular Size b) Solubility c) Interaction of the component with stationery
phase (column packing) d) Adsorption etc.
5.1.4. The separated components will be eluted in an order. The elution time is specific for a
particular component, whenever similar test parameters are applied. Every component
eluted will be identified by the detector as a series of electric signals and it will be
communicated to the computer.
5.1.5. The computer software converts & represents these signals with peaks in the
chromatogram.
5.2. Precautions:
5.2.1. Before executing a sample run, ensure the following:
5.2.2. Confirm that suitable column is installed.
5.2.3. Confirm that the Liquid chromatography system is stabilized. (There should not
be

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any Ghost peaks or Noise).

5.2.4. Prepare the sample solution as per method in clean glass ware.
5.2.5. Syringe used for injection is Clean.
5.2.6. Before injecting a different sample, clean the injector loop and syringe.
5.2.7. Confirm that mobile phase required for Sample run is sufficient and there are no
bubbles present in the suction pipeline (or at suction filter).
5.3. Control of HLC system:
5.3.1. Access control to HPLC system is necessitated to have better control on the use of
instrument and to protect the integrity of analytical data generated as per regulatory
requirements.
5.3.2. Users of HPLC system shall be classified in two categories as given below.
 Administrator
 Analysis
5.3.3. Quality Control Manager shall be the Administrator and the identified analysts for
Operating HPLC system shall be the operators of the instrument.
5.3.4. User Name and Password shall be created for both Administrator and analysts for
documented in the annexure-IV. The passwords shall be maintained secret and not
disclosed to each other.
5.3.5. User name and pass word shall be unique and shall not be repeated to newer persons
in case of resignations of existing personnel. In such cases, they shall be made as
obsolete and documented.
5.3.6. Administrator shall have all the powers.
 To load the method.

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 To modify the method as per requirement.

 To make minor adjustments in the existing method for better resolution of peaks.
 To modify shall the integration parameter as per requirement to get accurate
results.
5.3.7. Analyst shall not have any power as that of Administrator and he shall only carry out
analysis as per the method stored. In the case of any problem, Analyst shall inform
Administrator for necessary support.
5.3.8. Audit trail should be reviewed as per the checklist.
5.4. Operation:
5.4.1. Switch on the HPLC quaternary pump and required detector (either Refractive index
Detector or Variable Ultraviolet Wave Length Detector) Column Compartment,
Auto Liquid Sampler.
5.4.2. Now switch on the computer and double click on Open Lab icon to open software.
5.4.3. Open Lab solution launcher is displayed. Now click on “Launch” to create new
methods, open existing methods and run samples on existing methods.
5.4.4. Select postrun analysis to view, integrate and print the chromatograms already
completed.
5.4.5. The Launch window is opened. The Instrument Wizard window consists of the
following windows.
a) Create new method b) Modify the method c) Modify the Method on disk
d) Create sequence d) Intelligent Wizard with window f) Run one sample
g) Run a sequence of samples.
5.4.6. Select Method view of the data explorer.

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5.4.7. Open the required method to run the sample, if the method is already existing.
Otherwise, create a new method with required test parameters, give a file name and
save it.
5.4.8. Now the method will be opened. Click “Download” tab to apply the test parameters
to the 1260 Infinity Agilent HPLC
5.4.9. “IDLE” status will appear in the chromatogram window and green indicator glows at
Program run signal of the L.C. system.
5.4.10. Now click on Assistant bar Control  “Single Run” or choose
Sequence Run to enter below mentioned sample details.
a) Sample ID b) Method (batch / Lot no) c) data file name (should be unique
to each sample run) and d) Result Path.
5.4.11. Enter all the above mentioned details and click “Start”.
5.4.12. Now the sample details are temporarily stored in the computer memory and the
instrument control is transferred from computer to the instrument (1260 Infinity
Agilent).
5.4.13. The analysis will be started and chromatogram is continued till the completion of run
time.
5.4.14. After completion of runtime the system status is changed to “Ready”.
5.4.15. Then the chromatogram obtained will be consisting of raw data only. The
chromatogram is to be integrated to give the results of the analysis.
5.4.16. If there is any similar sample for analysis, perform the analysis as explained above.
Otherwise give washing to the column with major solvent used in the mobile phase.

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5.4.17. If any buffers are used in the preparation of the mobile phase, give Column
washing with water first and then wash with major solvent used in the mobile phase.
5.5. Integration Of The Chromatogram:
5.5.1. Click on the “Post Run” icon of the L.C. solution Launcher window. Now select the
desired chromatogram file from data file view of the data explorer.
5.5.2. Double click the file icon to open the chromatogram.
5.5.3. Choose the data analysis view of the chromatogram window.
5.5.4. Now open Manual Integration bar by either selecting the icon (on tool bar) or select
View  Manual Integration bar from standard menu.
5.5.5. Select all the peaks and remove the negligible peaks from the chromatogram.
Remove the peaks that already exist in the blank run.
5.5.6. Select all the unnecessary rows of the integration table and delete them. Now click
on “Save “icon.
5.5.7. The integration is now completed.
5.6. Printing The Chromatogram:
5.6.1. Select and open the desired Report Layout (Template which is already designed) from
reports view of data explorer of the POST RUN.
5.6.2. Now switch to data file view of the data explorer.
5.6.3. Select the desired chromatogram by single click. Now click and drag the
chromatogram icon on to the Report Layout (Template).
5.6.4. The report layout will appear in the standard format. Now verify the report and take
print out.

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5.7. Maintenance Instructions:

5.7.1. Perform the Column washing of the H.P.L.C. system daily once and also while
switching between different types of samples.
5.7.2. Clean the Syringe, Glass ware and Injector Loop thoroughly while analyzing different
type of samples.
5.7.3. Rinse the Syringe with the solvent used for sample preparation, before injecting the
sample every time through soft ware.
5.7.4. Monthly once remove the column, fix “UNION” and give washing to the entire
HPLC system with IPA for 3 to 4 hours.
5.7.5. In case of computer system hang-up/ disconnection with L.C., let the sample run
become complete. Now open the L.C. solutions software, the analysis data will be
transferred to another computer (By data recovery of the software).
5.7.6. Record every maintenance activity in the instrument log book, including washings of
the system.
6.0 Related Documents:
6.1 Instrument Log Book for HPLC. Annexure-I
6.2 HPLC Column Log Book LOG, Annexure II
6.3 List of HPLC columns Annexure III
6.4 Access control of HPLC system record-Annexure-IV

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7.0 Revision Record:

Date Revision Status Details of Revision Remarks
01-10-2011 00 First issue --

01-10-2014 01 Periodical Updation --
20-10-2014 02 To incorporate control of HPLC Under point No.5.3
system
03 Periodical Updation --
04 Incorporation -Audit trail should be Under point No.5.3.8
reviewed as per the checklist.

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023 Operation Procedure For High Pressure Liquid Chromatography (HPLC) - 04

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SRI VYJAYANTHI LABS PVT. LTD.

PLOT NO. 27/A, JNPC, PARAWADA (M) VISAKHAPATNAM – 531 019.

Doc No.: SVLL/QC/SOP/023-04

Prepared by Reviewed by Approved by

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Doc No.: SVLL/QC/SOP/023-04 Page 2 of 8

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any Ghost peaks or Noise).

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 To modify the method as per requirement.

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5.7. Maintenance Instructions:

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7.0 Revision Record:

01-10-2011 00 First issue --

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