1) Purpose : The purpose of this document is to provide calibration procedure for

High Performance Liquid Chromatography

2) Scope : The scope of this document is to provides calibration
Procedure for High Performance Liquid Chromatography
Instrument NO: ADTL/INS/ QC/001
3) Responsibility : Q.C. Officer

4) Accountability: Q.C. Manager

5) Precautions : ---

6) Procedure:

6.1 FOR PUMP

1. FLOW RATE

ACCURACY

1. Select the water as mobile phase and temperature at 25°C.

2. Put the suction filter into the reservoir containing the water as a

mobile phase.

3. Set the flow rate as 2 mL/min and open the bypass system

4. And start purging to remove air bubble from the tubing’s. Decrease

the flow rate slowly and stop the pump.

5. Connect the union between pump outlet and

6. Collect the mobile phase at the detector outlet into a cleaned

dried 10 mL measuring cylinder (previously weighed) for

known time i.e. at 0.5mL/min for 20minutes, 1.0 mL/min for 10

minutes and 1.5 mL/min for minutes 6.6 min and calculate the

flow rate per minute by given formulae

Volume dispensed in the selected time

 = wt. Of filled measuring cylinder (w2) – wt.of empty measuring cylinder (w1)

Wt./mL of water at 25°C

i. Repeat the above procedure for flow rate of 0.5mL/min for 20 min , 1.0 mL/
min for 10 min and 1.5 mL/min for 6.66 minutes.
ii. Record the observation

2. INJECTION LINEARITY:

Chromatographic conditions:
Mobile phase: Water: Methanol (30: 70)
Flow rate: 1 mL/min
Injection volume: 20 µL
Run time: 5 min
Column temperature: Ambient
Wavelength: 266 nm
Column ID:
Balance ID:

Caffeine solution preparation (0.01mg/mL): Transferred ____________ mg of caffeine into 100 mL

volumetric flask, dissolved and diluted up to mark with mobile phase. Further dilute 10 mL in to 100
mL with mobile phase.

i. Run the system as per the above chromatographic condition. Weight until the

baseline stabilizes, then, inject 10µl of methanol as a blank.

ii. Inject 10µL, 20µL,30µL,40µL and 50µL of the test sample in duplicate.

iii. Record the observation as per given in Annexure II. Plot the graph of injection

volume Vs. Response (peak area).

3. INJECTION PRECISSION:

Chromatographic conditions:
Mobile phase: Water: Methanol (30: 70)
Flow rate: 1mL/min
Injection volume: 20 µL
Run time: 5 min
Column temperature: Ambient
 Wavelength: 266 nm
Column ID:
Balance ID:

Caffeine solution preparation (0.01mg/mL): Transferred ____________ mg of caffeine into 100 mL

volumetric flask, dissolved and diluted up to mark with mobile phase. Further dilute 10 mL in to 100
mL with mobile phase.

i. Run the system as per the above chromatographic condition. Weight until the

baseline stabilizes, then, inject 10µl of methanol as a blank.

ii. Inject 10µL,20µL,30µL,40µL and 50µL of the test sample in duplicate.

iii. Record the observation as per given in Annexure II.

FOR DETECTOR:

4. DETECTOR LINEARITY:

Chromatographic conditions:
1. Mobile phase: Water: Methanol (30: 70)
2. Flow rate: 1ml/min
3. Injection volume: 20 µL
4. Run time: 5 min
5. Column temperature: Ambient
6. Wavelength: 266 nm
7. Column ID:
8. Balance ID:
Preparation of standard stock solution: Weighed about ___________ mg of caffeine in to
________________ mL volumetric flask. Dissolved and diluted with mobile phase.

i. Prepared the test sample of Caffeine 10 µg/mL to 50 µg/mL at the interval of 10

µg/mL in methanol
ii. Run the system as per above chromatographic condition, wait until the baseline
stabilizes, then, inject 20µL methanol as blank.
iii. Inject 20µL of test sample in duplicate at the wavelength of 266 nm.
 iv. Separately inject each 10 µg/mL to 50 µg/mL solution in duplicate and take the
mean of the solution for linearity study.
v. Plot the graph for conc. Vs. Response (peak area) & calculate the coeffient of
regression.
vi. Record the observation

5. WAVELENGTH ACCURACY

Chromatographic conditions:
1. Mobile phase: Water: Methanol (30: 70)
2. Flow rate: 1mL/min
3. Injection volume: 20 µL
4. Run time: 5 min
5. Column temperature: Ambient
6. Wavelength: 266 nm
7. Column ID:
8. Balance ID:
Preparation of standard stock solution: Weighed about ___________ mg of caffeine in to
________________ mL volumetric flask. Dissolved and diluted with mobile phase.

i. Run the system as per above chromatographic condition, wait until the baseline
stabilizes, then, inject 20µL methanol as blank.
ii. Inject 20µL of test sample in duplicate at the wavelength of 266 nm.
iii. Repeat the process at the wavelength of 261 nm to 271 nm at the interval of 1
nm.
iv. Process and save the acquired data and record the response at each
wavelength in calibration
v. Compare the results and measure the wavelength and max. in terms of max.
Response of area.

FOR COLUMN OVEN

6. OVEN TEMPERATURE

i. Set the column oven to 25°C.

ii. Place the probe of the thermometer inside the column oven.

iii. Note down the initial temperature displayed on the instrument.

 iv. Note down the temperature displayed by the probe.

v. Allow the column oven to stand for 30 min. And note down the temp.

vi. Repeat the above procedure similarly for temp. 25°C, 40°C, 50°C and 60°C.

vii. Record the instrument displayed temp. and thermometer reading

viii. Enter the details in Annexure-II.

7. SAMPLE COOLER TEMPERATURE CHECK

i. Set the sample cooler temperature to 25°C.

ii. Place the probe of the thermometer inside the sample chamber.

iii. Note down the initial temperature displayed on the instrument.

iv. Note down the temperature displayed by the probe.

v. Allow the sample chamber temperature to stand for 30 min. And note down the

temp.

vi. Repeat the above procedure similarly for temp. 5°C, 15°C and 25°C.

vii. Record the instrument displayed temp. and thermometer reading

viii. Enter the details in Annexure-II.

 Adichunchanagiri Drug testing Lab Doc No. : Revision No.: 00
ADTL/INS/DTG/005
Standard Operating Procedure Supersedes : Nil Effective Date :
28.08.2020

Disintegration Test Apparatus. Review Date :27.08.2022 Page 7 of 8

7) Reporting : Q.C.Manager

8) Abbreviations : SOP = Standard Operating Procedure

QC = Quality Control
CAL = Calibration
OPR = Operation
NO = Number

9) Attachment : 9.1 Calibration format of High Performance Liquid

Chromatography
9.2 Attachment I [Records of Acceptance]
10) History :
RECORD OF SOP REVISION

Revision Month of Change Authorized

Reason for Revision
No. Revision proposed by by
000 New SOP -----
001 Change in format of SOP
002 Change in format and content
of SOP
003 Change in format and content
of SOP

11) Distribution : 11.1 Master copy to Q.A. Dept.

11.2 First Xerox copy to Q.C. Manager
11.3 Second Xerox copy to Q.C. officer.
11.4 Third Xerox copy to Display copy

Reviewed by Approved by Issued by

Name: Name: Name: Name:

Signature: Signature: Signature: Signature:

 Adichunchanagiri Drug testing Lab Doc No. : Revision No.: 00
ADTL/INS/DTG/005
Standard Operating Procedure Supersedes : Nil Effective Date :
28.08.2020

Disintegration Test Apparatus. Review Date :27.08.2022 Page 8 of 8

12) Note :

IN CASE OF ANY CHANGES IN THE SOP’S, THE CHANGES HAVE TO BE

APPROVED BY THE RESPECTIVE DEPARTMENTAL HEADS AND
AUTHORISED BY Q.A. MANAGER PRIOR TO THEIR IMPLEMENTATION.

Reviewed by Approved by Issued by

Name: Name: Name: Name:

Signature: Signature: Signature: Signature: