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

SOP for testing of ETP water

sop for Calibration and Maintenance of Laboratory Instruments and Equipment

Disposal of Residual Sample or Left Over Material

sop for for Laboratory Incident

standard operating procedure temperature monitoring

sop for drying of silica gel

sop for operation of infrared moisture balance

sop for preparation of mobile phase

sop for treated RO water

sop for Preparation and Issuance of Analysis protocol standard

sop of placebo and impurity stock solutions