HPLC Calibration Procedure

Check HPLC chromatography (Pump) for the following:
I. Checkpoint: Leakage test (By Pressure Drop)
II. Flow rate calibration

CHECKPOINT: 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 into the Water HPLC grade through a suction filter.

3. Allow the 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 the Calibration Log.
7. Make entry of the HOLC column usage in the Column Usage Log Register.
8. Make entry of the user into the Instrument Usage Log Register.
9. Compare the result for its compliance with the 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 the 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 the calibration log.
6. Repeat the procedure for 1.0 ml, 1.5 ml and 2.0 ml/ min. flow rates.
7. Repeat step 3 to 6 but using methanol HPLC grade as mobile phase instead of water.
8. Compare the results for its compliance with limits given in the Calibration Log and put the remark regarding the Calibration Status.
9. Make entry of the usage of the instrument and HPLC 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

Solution Mixer Preparation

1. Take clean and dry 50 ml volumetric flask.
2. Pipette out 1.0 ml of Benzene and Toluene into the clean and dry 50 ml volumetric flask.
3. Make up the volume to 50 ml with Methanol and mix well.

Chromatographic Condition

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

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 µl in triplicate and record the chromatograms.
4. Repeat the injection of the above solution by injecting 15, 20, 25, 30 µl 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 HPLC column in the Instrument Usage Log Register and Column Usage Log Register respectively.
8. Plot the curve for the 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 with the 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.

CHECKPOINT: D2 LAMP ENERGY CHECK (DETECTOR)

1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed.
2. On the Detector’s 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.
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 with the 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 into the clean, dry 50 ml volumetric flask separately.
3. Make up the volume to 50 ml with Methanol and mix well.

Solution (Level)	ml of Benzene to be taken	Ml of Toluene to be taken	To be Diluted with Methanol 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 Equipment’s Parameter Setup Procedure.
HPLC 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 µl

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.
6. Make entry of the usage of the instrument and HPLC 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.  Checkpoint  : Leakage Check Test (Pressure Drop)
P.Max to be set	P.Max Set		Start Time		Stop Time		Total Time		Pressure Observed		Remark
300 bar
B.  Flow Rate calibration :
Flow Rate (ml / min.)		Time required to collect 10 ml mobile phase						Limit		Remarks
		Theoretical (in sec.)		Actual with water		Actual with Methanol
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

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)
HPLC Column	ODS C18, (25 cm x 4.6 mm ID, 5 µm)
Flow Rate	1.0 ml / min.
Wavelength	254 nm

Injection volume		Injection -1		Injection -2		Injection -3		Mean	% RSD : (Not more than 2.0%)
		Benzene	Toluene	Benzene	Toluene	Benzene	Toluene
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 :
Wavelength to be set	Wavelength Set	Reference Energy Observed	Limit
254 nm			Not less than 200

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)
HPLC Column	ODS C18, (25 cm x 4.6 mm ID, 5 µm)
Flow Rate	1.0 ml / min.
Wavelength	254 nm
Range	1.0 AUFS
Injection Volume	20 µl

Solution (Level)	Benzene Area		Toluene Area		Area Ratio (Benzene/Toluene)
	RT	Area	RT	Area
1
Mean :
% RSD  :		---		---
2
Mean :
% RSD  :
3
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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