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sop for Operation and Calibration of High Performance Liquid Chromatography

sop for Operation and Calibration of High Performance Liquid Chromatography

 

1.1 OBJECTIVE
1.0 To lay down the procedure for Operation and Calibration of High Performance Liquid Chromatography.
2.0 SCOPE
2.1 This SOP is applicable for the Operation and Calibration of HPLC

3.0 RESPONSIBILITY
3.1 QC Officer or QC Executive
4.0 ACCOUNTABILITY
4.1 QC Manager
5.0 Procedure
5.1 Operation Procedure
5.1.1 Check that the power supply to the instrument is switched “OFF” before cleaning.
5.1.2 Clean the instrument with a clean dry cloth and change the water of rinsing vial at every day.
5.1.3 Open the main switch of the power and sequentially “ON” the power of the instrument from the main panel and enter the password < 00000 >, press to ok.
5.1.4 Connect the suitable solvents Initiate purge of the PUMP by pressing purge key on the main panel.
5.1.5 Initiate purge of the Auto sampler by pressing purge key on the main panel.
5.1.6 Rinse the Auto sampler by pressing rinse key on the main panel.
5.1.7 To operate the Software, switch on the power of all Liquid Chromatographic (LC) units and System Controller.
5.1.8 Switch on the power of Computer, Monitor and Printer.
5.1.9 Click on open lab CDS software icon on the desktop the main menu will be opened.
5.1.10 Purge all the ports individually with 100% methanol at the flow rate of 5 ml/min
5.1.11 Flush the system using union for about 15 min with 100% Methanol at the flow rate of 3 ml/min from all the ports respectively.
5.1.12 Purge all the ports individually with hot water for about 5 minutes each at the flow rate of 5 ml/min.
5.1.13 Finish the system using union for about 15 minutes with hot water at the flow rate of 3ml/min from all the ports respectively.
5.2 Calibration Procedure
5.2.1 Accuracy of flow rate
5.2.1.1 Set the flow rate to 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml and 5.0 ml and record the volumes of solvent collected in 5
minutes five times as per Annexure -1

                               Calculation of flow rate = Collected water weight

                                                                            Time X 0.997

(Where.: 0.997 is density of water at 25oC)
5.2.2 Accuracy of Injection Volume:
5.2.2.1 Purge the injector system with 100% water to ensure the complete washing of injector.
5.2.2.2 Fill a vial with water for injection and note down the weight (W 1). Place the vial in tray no.-1 and at no.1 position.
5.2.2.3 Set the instrument parameters as given below:
Mobile phase: A mixture of methanol and distilled water in the proportion of 80: 20 (% v/v)
Column: 250 X 4.6 mm, C 18
Flow: 1.0 ml/min
Detector wave length: 254 nm
Injection volume: 20 µl
Run Time: 1.0 Minutes
5.2.2.4 Run the sequence of 36 injections with above given parameters, after completion of sequence take out the
vial from the tray and weight it again (W 2). Calculation of Injection volume is as given below. Record the
result as per log book

Injection volume =       (W 1) – (W 2) X1000/No. of Injections (36) 

 

Acceptance Criteria: The Injection volume accuracy will be within +0.4 μL
5.2.3 Carry-Over Test
Chromatographic Condition
Mobile phase: A mixture of methanol and distilled water in the Proportion of 80: 20 (% v/v)
Column: 250 X 4.6 mm, C 18
Flow rate: 1.0 ml/min
Detector wavelength: 254 nm
Injection volume: 20 µl
Preparation of Solution: Accurate weigh 50 mg of Caffeine in 100 ml volumetric flask. Dilute it with
methanol and make up to 100 ml. Mix well.
Inject single injection of the above solution (500 ppm) of Caffeine followed by single blank run.

Calculate the area count of the standard and of blank at the same retention time. Record the result as per log book
Percentage area of the blank w.r.t. standard

Area of Blank    X    100     X   Purity of caffeine      

Area of Standard   X     100

Acceptance Criteria: The percentage of the area count of the blank with respect to the standard should not be more than 0.01%.

5.2.4 Precision of Injection:
Chromatographic Condition:
Mobile phase : A mixture of methanol and distilled water in the Proportion of 80: 20 (% v/v)
Column: 250 X 4.6 mm, C 18
Flow rate: 1.0 ml/min
Detector wavelength: 254 nm
Injection volume: 20 µl
Preparation of Solution: Accurate weigh 100 mg of Caffeine in 100 ml volumetric flask. Dilute it with methanol and

make up to 100 ml (Solution A). Mix well. Pipette out 10 ml from solution A in 100 ml volumetric flask and make up the volume up to 100 ml (Solution-B)
Inject 5 consecutive injections of the solution-B (100 ppm) of Caffeine and record the chromatograms as per Annexure-4.

Calculate the relative standard deviation of area counts.
Acceptance Criteria: The RSD of the response of 5 consecutive injections of the sample solution-B should not be more than 1.0%.
5.2.5 Linearity of Auto injector
5.2.5.1 Inject triplicate injection of 5 different volume i.e. 5 µl, 10 µl, 15 µl, 20 µl and 25 µl of solution-B and record

the chromatograms as per log book. Plot the graph of area response against injection volume.
Acceptance Criteria: The area responses produced by the caffeine solution should be linear and correlation coefficient should be NLT 0.999
5.2.6 Wavelength Accuracy of Detector
5.2.6.1 Procedure: Inject 20µl of Caffeine Solution -B as prepared in precision, 3 times. Each time set the detector at following

different wavelengths between 201 to 210 nm, 241 to 250 nm 268 to 278 nm. Record the areas of the responses of Caffeine as per log book
Acceptance Criteria: The detector shows maximum peak area at 201 to 210 is 205 ±1 nm, at 241 to 250 nm is 245 ± 1 nm and 268 to 278 nm is 273 ± 2 nm.

5.2.7 Linearity of Response
5.2.7.1 Preparation of stock solution: Accurately weigh 100 mg of Caffeine in a 100 ml of volumetric flask.

Dissolve and make up the volume to 100 ml with methanol (Solution-A). Repeat a series of solutions having following

concentrations by diluting the stock solution.
Solution Concentration Dilution
Solution 1 50 ppm 5ml stock soln. —— 100 ml with methanol
Solution 2 100 ppm 10 ml stock soln. —— 100 ml with methanol
Solution 3 150 ppm 15 ml stock soln. ——- 100 ml with methanol
Solution 4 200 ppm 20 ml stock soln. —— 100 ml with methanol
Solution 5 250 ppm 25 ml stock soln. ——- 100 ml with methanol
Inject about 5 solutions separately to obtain chromatograms and record the areas of Caffeine responses as per Annexure-7.

Plot the graph of area response against concentration.
Acceptance Criteria: The area responses of Caffeine Solution should be linear and correlation coefficient should be NLT 0.999
5.3 Frequency of Calibration:
5.3.1 Calibration shall be done half yearly + 1 week.
6.0 ABBREVIATIONS

Abbreviation used
SOP          Standard Operating Procedure
HPLC      Calibration of High Performance Liquid Chromatography
QC          Quality Control

 

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sop for preparation of mobile phase

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sop for Preparation and Issuance of Analysis protocol standard

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difference between UPLC and HPLC

sop for for Emergency Eyewash and Shower

sop for operation and calibration of total organic carbon analyzers

sop for operation of cobb tester

sop for Operation and calibration of atomic absorption spectrophotometer

sop for Operation and calibration of gas liquid chromatograph

sop for operation of humidity oven

sop for operation and calibration of serological water bath

sop for monitoring of drain trap

sop for destruction of analytical samples after testing and control samples

sop for destruction of used chemicals

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sop for Operation and calibration uv cabinet

sop for Operation and calibration of bulk density apparatus

sop for operation and calibration of shore hardness tester

sop for operation of rub proofness tester

sop for monitoring of purified water

sop for Retesting of packaging materials

sop for Retesting and resampling of raw materials

sop for Control of issuance of record of analysis green sheets

sop for Control of computer passwords

sop for sampling of packaging materials PM

sop for sampling of sterile raw material

sop for sampling of intermediates and finished products

sop for operation and calibration of friability test apparatus

sop for approval and rejection of packaging materials

sop for non conformance of RM PM and finished product

sop for collection storage and disposal of control samples

sop for trend analysis of finished products

sop for Chromatographic practices and system suitability

SOP For Good Laboratory Practices

sop for cleaning and operation of sieve shaker

general specification of packing material cartons

sop for Password for Analytical Instrument and LIMS software

sop for Rounding off numerical analytical results

sop for sampling of bulk and finished product

sop for cleaning of spillage material

sop for Handling of Reference Standard

sop for hplc column maintenance and washing

procedure for sampling and handling of bulk sample

STP for borewell generation point (raw water storage tank)

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validation of volumetric solution 0.1m ammonium thiocyanate

handling of reference standard and preparation of working standard

sop for water sampling and analysis

sop for operation for validation of excel worksheets

sop for laboratory cleaning

sop for stability of volumetric solutions

sop for preparation of raw material in process finish product packing material data sheets

sop for handling of hazardous chemicals

sop for handling of glassware and allocation of identification number

sop for operation cleaning and calibration of bursting strength tester

sop for rounding off the analytical test results

procedure for Analyst Qualification

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procedure for maintenance of desiccators

sop for for hplc column receipt checking id no and regeneration

safety data sheet for laboratory chemicals

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procedure for disposal of expired chemicals, reagents and solvents

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sop for preparation and standardization 0.1M Disodium Edetate

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sop for Operation and Calibration of High Performance Liquid Chromatography

sop for UV & Visible Spectrophotometer

procedure for Cleaning of laboratory glassware

Cleaning of Instrument, Instrument bench and surrounding area of Quality

Safety Precaution in Quality Control Department

Operation & Calibration of Analytical Balance

Calibration of Glassware in Quality Control Department

handling of samples received in Quality Control

Cleaning and Operation of Refrigerator

Operation, Cleaning and Calibration of water bath

Operation & Calibration of Refractometer

Operation and Cleaning of Centrifuge Apparatus

cleaning, operation & calibration of Vernier caliper

Calibration of Fourier Transform Infrared Spectrophotometer (FTIR)

Cleaning and operation of Moisture Analyzer

Cleaning & Operation of Vacuum pump in Quality Control Department

Operation and Calibration of Polarimeter

Cleaning and operation of Magnetic Stirrer

Cleaning Operation and Calibration of Melting Point

Operation Cleaning and Calibration of Muffle Furnance in Quality Control Department

procedure of operation and Cleaning of Sonicator

Operation Cleaning & Calibration of pH meter in Quality Control Department

Entry and Exit in Quality Control Department

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