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Standard Operating Procedure HPLC

This document outlines the standard operating procedure for using high performance liquid chromatography (HPLC) to analyze acetylsalicylic acid production. It describes the critical steps, scope, responsibilities, required reagents and equipment, risk assessment, and detailed procedure for preparing the mobile phase, instrument, method, sequence, running the analysis, obtaining results, washing, and analyzing paracetamol product using HPLC.

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272 views

Standard Operating Procedure HPLC

This document outlines the standard operating procedure for using high performance liquid chromatography (HPLC) to analyze acetylsalicylic acid production. It describes the critical steps, scope, responsibilities, required reagents and equipment, risk assessment, and detailed procedure for preparing the mobile phase, instrument, method, sequence, running the analysis, obtaining results, washing, and analyzing paracetamol product using HPLC.

Uploaded by

h9g886qdnp
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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SOP: High Performance Liquid Chromatography

This Standard Operation Procedure outlines the process of HPLC as a method in relation to the quantitative
determination of acetylsalicylic acid during its’ production.

1.0 Purpose:

1.1 To confirm the identity & analyse the synthesized Paracetamol by High Performance
Liquid Chromatography as well as to document and ensure that Good Laboratory Practices
are in place.

2.0 Critical Points:


2.1 If there was phosphate in the buffers, leave the contaminated pump running at
0.05 mL/min for the whole night with clean water. Phosphates should be used as
little as possible.
2.2 “Floshing the injector" is the process of cleaning the injector by adding the
washing solution to the sample loop and switching the port from load to inject.

3.0 Scope
This SOP is applicable to members operating in the Quality Control Department.

4.0 Responsibilities
Quality Control Supervisor

5.0 Required Reagents and Equipment:

5.1 Reagents

• Sodium Dihydrogen Phosphate


• Deionized water (solvent)
• Phosphoric Acid (If pH needs to be altered)
• Methanol
• Synthesized Paracetamol

5.2 Reagent Measurements Required:

• 2.04G Sodium Dihydrogen Phosphate


• 800ML Deionized Water
• 200ML Methanol
• 800ML Buffer Solution
• 80MG Synthesized Paracetamol

5.3 Equipment

• Analytical Balance, capable of accurately measuring to minimum


0.001grams.
• Glass Beaker capable of holding 1000ml
• Volumetric Flask, capable of holding 100 ml & 1000ml
• 0.45 Filter Paper
• Petri dishes (weighed)
• HPLC Instrumentation

6.0 Risk Assessment


6.1 see attachment 1 on risk assessment for chemical and equipment safety.

6.2 gloves, lab safety goggles and laboratory coat are required for the duration of this
procedure.

7.0 Procedure:

7.1 Mobile Phase Preparation

7.1.1 Weight out 2.04g of sodium hydrogen phosphate on the analytical balance.

7.1.2 In a glass beaker dissolve the weighted amount of sodium dihydrogen phosphate
in 800ml of deionized water.
7.1.3 Adjust the pH of the solution to a pH 4.8 using phosphoric Acid/Sodium
Hydroxide (NaOH) if needed.
7.1.4 Make this solution up to 1000ml using a volumetric flask, making it up to the
mark using deionized water & labelling as ‘buffer solution’.
7.1.5 In a separate container, add 200ml of methanol to 800ml buffer solution & mix
well.
7.1.6 Filter this solution through a 0.45 filter paper.
7.2 Stock Solution of Paracetamol Preparation
7.2.1 Weight out 80mg of paracetamol on the analytical balance.

7.2.2 Dissolve this sample in the smallest amount of methanol possible.

7.2.3 Dilute this solution to the mark with deionized water in a 100ml volumetric

7.3 HPLC Instrument Set Up

7.3.1 Weight Power up the computer and log on.

7.3.2 Switch on the HP35900 dual channel interface.

7.3.3 Wait for the beep.

7.3.4 Switch on the HP1100 degasser, Quat pump, VWD, COLCOM, ALS & ALS
thermostat systems.

7.3.5 Double click on the “INSTRUMENT 1 ONLINE” icon.

7.3.6 Wait for the “METHOD & RUN CONTROL WINDOW” to open.

7.3.7 Allow for a graphical system diagram to appear on the middle right of the screen
with each component of the process represented by an icon.

7.3.8 The system is colour coded to determine the status of each component.

7.3.9 The Select Instrument/System on icon from the top of the screen

7.3.10 Click “ALS” icon & select "THERMOSTAT” & switch off.
7.3.11 Click on the column icon & select “COLUMN THERMOSTAT METHOD” & select
“NOT CONTROLLED”

7.3.12 Click the “VWD” icon & select “Control/ON/OK”


7.3.13 Click on the pump icon & select “SET UP PUMP”
7.3.14 Open the purge valve tank links to the solvent being used.
7.3.15 Set the Flow rate to 5.000ml/min & choose “OK”.
7.3.16 Allow the mobile phase to pump through the system for 20 mins. to purge the
instrument pup of any air bubbles present.
7.3.17 Set the volume of the mobile phase in the solvent reservoir.
7.4 HPLC Method Set Up
7.4.1 Click on “METHOD”.

7.4.2 Select “NEW METHOD”.

7.4.3 Click on “METHOD”.

7.4.4 Click on “EDIT ENTIRE METHOD” and activate all four boxes.

7.4.5 Click “OK”.

7.4.6 Click on text area in the Method Comments section & type in “Analysis of
Paracetamol”.
7.4.7 Click “OK”.

7.4.8 The Instrument & Acquisitions section appears.

7.4.9 In the pump dialog box fill in the parameters for the instrument:

• Set the Flow Rate to 1.5ml/min.


• Set the run time to 10 minutes.
• Set the solvent channels for line A containing mobile phase.
• Set maximum pressure to 400 bar & the minimum pressure to 0 bar.

7.4.10 Set up the injector, fill the injector volume.

• Set the injection to 20ml.

7.4.11 The ADC1 ADC Signal Source dialog box appears.

7.4.12 Click “OK”.

7.4.13 The ACD1 Timed Extent Table: Instrument 1 dialog box appears.

• Set the wavelength of the instrument to 254nm.


7.4.14 Click “OK”.

7.4.15 Signal Details dialog box appears.

7.4.16 Click “OK”.


7.4.17 Edit Intergration Events box appears.

7.4.18 Click “OK”.

7.4.19 Specify the printer.

7.4.20 Click “OK”.

7.4.21 Instrument curves box appears.

7.4.22 Click “OK”.

7.4.23 C Runtime Checklist appears tick Data Acquisition & Standard Data.

7.4.24 Click “OK”.

7.4.25 Save method by clicking “METHOD SAVE AS” type in the method name & click “OK.”

7.5 HPLC Sequence Set Up


7.5.1 Click on “SEQUENCE.”

7.5.2 Select “SEQUENCE TABLE”

7.5.3 Full sequence table is required.

7.5.4 Click on “INSERT/ FILL DOWN WIZARD.”

7.5.5 Into the table with the number position for each sample being red enter;

• Sample Name
• Method Name
• Sample Type
• No. Of Injections

7.5.6 Ensure that the samples are in the correct positions according to the tabel information
for the instrument.
7.5.7 Click “OK”

7.5.8 Select SEQUENCE TABLE/ SAVE SEQUENCE TABLE AS” & type in a name like that of the
method.
7.5.9 Select “SEQUENCE PARAMETERS.”

7.5.10 Data file box appears, enter a name in the Subdirectory text box.

7.5.11 In the operator text box enters group names.

7.5.12 Click “OK.”

7.5.13 Click on “SAVE SEQUENCE TABLE.”


7.6 Starting the Run on HPLC
7.6.1. Load method.

7.6.2 Load Sequence.

7.6.3 Select “RUN CONTROL/ RUN SEQUENCE.”

7.7 Obtaining Results on HPLC


7.7.1 HPLC Chromograms are printed for each sample run with the peak retention times
labelled
7.7.2 Once all the data is gathered, shut off the instrument by selecting
“INSTRUMENT/SYSTEM OFF.”

7.8 Washing on HPLC


7.8.1 Clean the injector equipment and column after use HPLC.

7.8.2 Wash with the washing solution (10% MeOH/H2O) to eliminate any bases, buffer salts,
or acids if you're using aqueous buffers.

7.9 Analysis of Product:

7.9.1 Complete the following Paracetamol analysis by High Performance Liquid


Chromatography analysis.
8.0 References

• www.pharmaguideline.com. (n.d.). SOP for HPLC Analysis and Documentation :


Pharmaceutical Guidelines. [online] Available at:
https://www.pharmaguideline.com/2011/02/sop-for-hplc-analysis-and-
documentation.html. {Accessed on 5th Nov 2023}
• Kam, R.K.-T., Chan, M.H.-M., Wong, H.-T., Ghose, A., Dondorp, A.M., Plewes, K. and
Tarning, J. (2018). Quantitation of paracetamol by liquid chromatography–mass
spectrometry in human plasma in support of clinical trial. Future Science OA, 4(8),
p.FSO331. doi: https://doi.org/10.4155/fsoa-2018-0039. {Accessed on 5th Nov 2023}
•
• HPLC Standard Operating Procedure. (n.d.). Available at:
https://s3.wp.wsu.edu/uploads/sites/33/2013/10/HPLC-Standard-Operation-
Guidelines.pdf. {Accessed on 5th Nov 2023}

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