HPLC Calibration Procedure : Pharmaceutical Guidelines

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HPLC Calibration Procedure

Know the procedure to calibrate the High Performance Liquid Chromatography ( HPLC) including leakage test, flow rate,
reproducibility and linearity, lamp energy and pump pressure drop in Pharmaceutical Quality Control.

Ankur Choudhary Print

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3 comments

Check HPLC chromatography (Pump) for the following :

I. Check point : Leakage test (By Pressure Drop)

II. Flow rate calibration

CHECK POINT : LEAKAGE TEST (BY PRESSURE DROP)

1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed.

2. Place inlet tubing of the Pump in to the Water HPLC grade through suction filter.

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HPLC Calibration Procedure : Pharmaceutical Guidelines

3. Allow mobile phase to flow for about 5 min.

4. Block Pump outlet with the block screw.

5. The pressure rises and on crossing the 300 bar, ERROR P-
MAX appears on the display window. Note the time. Press
CE key and observe the pressure drop for 5 min.

6. After 5 min., record the pressure in calibration Log.

7. Make entry of the column usage in the Column Usage Log
Register.

8. Make entry of the usage into the Instrument Usage Log
Register.

9. Compare the result for its compliance against limit given in the Calibration Log and put the remark
regarding the HPLC chromatography Calibration Status.

10. In case of non-compliance, follow the Maintenance Program.

Related: Principle and Working of HPLC Chromatography

FLOW RATE CALIBRATION

1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed.

2. Ensure that, the Pump is passing the Leakage Test (By Pressure Drop).

3. Keep the Drain tube in such a way that the mobile phase (Water) drops falls into 10 ml clean, dry
volumetric flask without touching the walls of the flask and start immediately the stopwatch when first drop
falls into the flask.

4. Wait till the collected mobile phase reaches 10 ml mark of the volumetric flask and Stop the stopwatch. .

5. Record the time required to collect the 10 ml mobile phase in calibration log.

6. Repeat the procedure for 1.0 ml, 1.5 ml and 2.0 ml/ min. flow rates.

7. Repeat the step 3 to 6 but using methanol HPLC grade as mobile phase instead of water.

8. Compare the results for its compliance against limits given in the Calibration Log and put the remark
regarding the Calibration Status.

9. Make entry of the usage of the instrument and chromatography column in the Instrument Usage Log
Register and Column Usage Log Register respectively.

10. Prepare Calibration Status Label and display on the instrument at the designated place.

11. In case of non-compliance, follow the Maintenance Program.

REPRODUCIBILITY AND LINEARITY OF INJECTION VOLUME

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HPLC Calibration Procedure : Pharmaceutical Guidelines

Solution Mixer Preparation :

1. Take clean and dry 50 ml volumetric flask.

2. Pipette out 1.0 ml of Benzene and Toluene in to the clean and dry 50 ml volumetric flask.

3. Make up the volume to 50 ml with Methanol and mix well.

Chromatographic Condition :

Column : ODS C18, (25 cm x 4.6 mm ID, 5 m)

Mobile Phase : Methanol : Water (70 : 30)

Flow Rate : 1.0 ml/min.

Wavelength : 254 nm

Injection Volume : 20 l

Calibration :

1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed.

2. Ensure that, the instrument is set according to the Chromatographic conditions.

3. Follow the Instrument Operating procedure, Inject 10 l in triplicate and record the chromatograms.

4. Repeat the injection of above solution by injecting 15, 20, 25, 30 l in triplicate.

5. Take the print out of the chromatograms and attach to the Calibration Log.

6. Record the Area and Retention times of the of the Benzene and Toluene peaks in the Calibration Log.

7. Make entry of the usage of the instrument and column in the Instrument Usage Log Register and Column
Usage Log Register respectively.

8. Plot the curve for area corresponding to Benzene to Toluene peaks v/s injection volume, Find out the
RSD (reproducibility) and record in the Calibration Log.

9. Find out the Correlation coefficient r2 for each peak at five levels and record in the Calibration Log.

10. Compare the result for its compliance against limit given in the Calibration Log and put the remark
regarding the Calibration Status.

11. Prepare Calibration Status Label and display on the instrument at the designated place.

12. In case of non-compliance, follow the Maintenance Program.

CHECK POINT : D2 LAMP ENERGY CHECK (DETECTOR)

1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed.

2. On the Detectors display window, some values at the functions l(nm), abs(AU), range(AUFS) and
lamp appears.

3. On the display, previously set value blinks at l(nm) function, enter the wavelength to 254 nm by pressing
numeric keys.

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HPLC Calibration Procedure : Pharmaceutical Guidelines

4. Press Func Back key and select lamp functions, enter 1 to select D2 lamp.

5. Further press Func Back key till REF EN appears.

6. Record the Reference Energy of the D2 Lamp at 254 nm in the Calibration Log.

7 Compare the result for its compliance against limit given in the Calibration Log and put the remark
regarding the Calibration Status.

8. In case of non-compliance, follow the Maintenance Program.

III. LINEARITY OF DETECTOR RESPONSE :

Solution Mixer Preparation :

1. Take three clean and dry 50 ml volumetric flasks.

2. Pipette out ml of Benzene and Toluene as per the following table in to the clean, dry 50 ml volumetric
flask separately.

3. Make up the volume to 50 ml with Methanol and mix well.
ml of Benzene to be taken Ml of Toluene to be taken To be Diluted with Methanol
Solution (Level)
to

1 0.5 1.0 50 ml

2 1.0 1.0 50 ml

3 1.5 1.0 50 ml

Chromatographic Condition :

1. Set each of the equipment to the following parameters as per the Equipments Parameter Setup
Procedure.

Column : ODS C18, (25 cm x 4.6 mm ID, 5 m)

Mobile Phase : Methanol : Water (70 : 30)

Flow Rate : 1.0 ml/min.

Wavelength : 254 nm

Injection Volume : 20 l

Calibration :

1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed.

2. Ensure that, the instrument is set according to the Chromatographic conditions.

3. Follow the Instrument Operating procedure, Inject each of the Solutions in triplicate and record the
chromatograms.

4. Take the print out of the chromatograms and attach to the Calibration Log.

5. Record the Area and Retention times of the of the Benzene and Toluene peaks in the Calibration Log.

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HPLC Calibration Procedure : Pharmaceutical Guidelines

6. Make entry of the usage of the instrument and column in the Instrument Usage Log Register and Column
Usage Log Register respectively.

7. Calculate the Area Ratio of Benzene to Toluene, Find out the Mean value of the ratios and record in the
Calibration Log.

8. Find out the Correlation coefficient r2 from the Mean area ratio values of the three levels.and record in
the Calibration Log.

8. Plot the curve for area ratio corresponding to Benzene to Toluene peaks v/s concentration, Find out the
RSD (reproducibility) and record in the Calibration Log.

9. Compare the result for its compliance against limit given in the Calibration Log and put the remark
regarding the Calibration Status.

10. Prepare Calibration Status Label and display on the instrument at the designated place.

11. In case of non-compliance, follow the Maintenance Program.

CALIBRATION OF HPLC ( Liquid Chromatography ) PUMP
Model No. : Make :

Instrument Code No. :

A. Check point : Leakage Check Test (Pressure Drop)

P.Max P.Max Set Start Time Stop Time Total Time Pressure Remark

to be set Observed

300 bar

B. Flow Rate calibration :

Flow Rate Time required to collect 10 ml mobile phase

Theoretical Actual with water Actual with Limit
Remarks
(ml / min.) Methanol
(in sec.)
0.5 ml 1200 1194 - 1206
1.0 ml 600 594 606
1.5 ml 450 443 457
2.0 ml 300 294 - 306

Calibration Status : Satisfactory / Not Satisfactory

Next Calibration Due :

Calibrated By : Checked By :

Date : Date :

CALIBRATION OF INJECTOR

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HPLC Calibration Procedure : Pharmaceutical Guidelines

Model : Make :

Instrument Code No. :
a. Preparation of solvent mixture :

Taken _____ ml (1.0 ml) of the ________ (Benzene) and ________ (Toluene) in to a _____ ml (50 ml)
clean, dry volumetric flask, make up the volume with methanol, mixed well.
b. Chromatographic Condition :
Parameters Test Condition Applied Condition
Mobile Phase Methanol : Water (70 : 30)

Column ODS C18, (25 cm x 4.6 mm ID, 5 m)

Flow Rate 1.0 ml / min.

Wavelength 254 nm

Injection volume Injection -1 Injection -2 Injection -3 % RSD :

Mean
(Not more than
2.0%)
Benzen Toluene Benzene Toluen Benzene Toluene
e e
10 ml RT
Area
15 ml RT
Area
20 ml RT
Area
25 ml RT
Area
30 ml RT
Area

Coefficient of co relations : r2 : _____________ (Limit : NLT : 0.999)

Calibration Status : Satisfactory / Not Satisfactory

Next Calibration Due :

Calibrated By : Checked By :

Date : Date :

CALIBRATION OF DETECTOR
Model : Make :

Instrument Code No. :

D2 LAMP ENERGY CHECK :

Limit
Wavelength to be set Wavelength Set Reference Energy Observed

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HPLC Calibration Procedure : Pharmaceutical Guidelines

Not less than 200

254 nm

LINEARITY OF DETECTOR RESPONSE :

a. Solution Preparation :
Solution ml of Benzene ml of Toluene in Methanol

(Level) To be taken Taken To be taken Taken To be diluted Diluted to

1 0.5 ml 1.0 ml 50 ml

2 1.0 ml 1.0 ml 50 ml

3 1.5 ml 1.0 ml 50 ml

Mobile Phase Preparation : Taken ______ ml of ___________ (Methanol HPLC grade) in to a _______ ml clean, dry volumetric
flask, added ____________ ml of ___________(Water HPLC grade), mixed well, allowed to cool to room temperature. Filtered
through 0.45/0.22 m membrane filter, degassed for _____ min. by vacuum / sonication.

Parameters Test Condition Applied Condition

Mobile Phase Methanol : Water ( 70 : 30)
Column ODS C18, (25 cm x 4.6 mm ID, 5 m)
Flow Rate 1.0 ml / min.
Wavelength 254 nm
Range 1.0 AUFS
Injection Volume 20 l

Solution Benzene Area Toluene Area Area Ratio

(Level) RT Area RT Area (Benzene/Toluene)
1


Mean :
% RSD : --- ---

2


Mean :
% RSD :

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HPLC Calibration Procedure : Pharmaceutical Guidelines

Mean :
% RSD :

Linearity of Ratio : Correlation Co-efficient r2 :

Limit : % RSD of the Retention Time : Not more than 2.0%
Linearity of Ratio : Correlation Co-efficient r2 : Not less than 0.999

Calibration Status : Satisfactory / Not Satisfactory

Next Calibration Due :
Calibrated By : Checked By :
Date : Date :

Related:SOP for Calibration of HPLC

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Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of
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3 comments:

Anonymous 29/6/11 11:17

many many thanks
Reply

Dipankar sarkar 26/8/15 18:43

perfect
Reply

Unknown 24/4/17 10:19

What is the difference between calibration and performance check?
Reply

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