HPLC Calibration SOP


HPLC Calibration SOP
1.0  OBJECTIVE:
To lay down the calibration procedure of High Performance Liquid Chromatography (HPLC) for reliable and accurate results.
2.0  SCOPE:
This shall be applicable to the installed High Performance Liquid Chromatography (HPLC) of Quality Control Instrument Laboratory.
3.0  RESPONSIBILITY:
3.1  Technical Assistant/ Helper.
3.2  Q.C Analyst.
4.0  ACCOUNTABILITY:
4.1  Manager-Quality Control.
5.0  MATERIAL AND EQUIPMENT:
5.1  High Performance Liquid Chromatography (HPLC).
5.2  Stopwatch.
5.3  Purified water.
5.4  Methanol.
5.5  Toluene.
5.6  Glassware.
6.0  PROCEDURE:
6.1  Make sure that the instrument is clean.
6.2  Switch on the HPLC apparatus and operate it according to operating procedure.
6.3  Calibrate the apparatus according to the instructions for the reliable and accurate results.
6.4  Calibration of pressure drop (Leakage test):
6.4.1         Ensure that, the instrument is ready for calibration and Start-up procedure is followed.
6.4.2        Place inlet tubing of the Pump in to the Water HPLC grade through suction filter.
6.4.3        Allow mobile phase to flow for about 5 min.
6.4.4        Block Pump outlet with the block screw.
6.4.5        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.
6.4.6        After 5 min., record the pressure in Calibration Log.
6.4.7        Make entry of the column usage in the Column Usage Log register.
6.4.8        Make entry of the usage into the Instrument Usage Log Register.
6.4.9        Compare the result for its compliance against limit given in the Calibration Log and put the remarks regarding the HPLC chromatography Calibration status.
6.4.10    In case of non-compliance, follow the Maintenance Program.
6.5  Calibration of flow rate:
6.5.1        Ensure that, the instrument is ready for calibration and Start-up procedure is followed.
6.5.2        Ensure that, the Pump is passing the “Leakage Test (By Pressure Drop)”.
6.5.3        Keep the Drain tube in such a way that the mobile phase (Water) drops falls into 10.0ml clean, dry volumetric flask without touching the walls of the flask and start immediately the stopwatch when first drop falls into the flask.
6.5.4        Wait till the collected mobile phase reaches 10 ml mark of the volumetric flask and Stop the stopwatch.
6.5.5        Record the time required to collect the 10 ml mobile phase in calibration log.
6.5.6        Repeat the procedure for 1.0ml, 1.5ml and 2.0ml/ min. flow rates.
6.5.7        Repeat the step 6.5.3 to 6.5.6 but using methanol HPLC grade as mobile phase instead of water.
6.5.8        Make entry of the column usage in the Column Usage Log register.
6.5.9        Make entry of the usage into the Instrument Usage Log Register.
6.5.10    Compare the result for its compliance against limit given in the Calibration Log and put the remarks regarding the HPLC chromatography Calibration status.
6.5.11    Prepare the calibration status Label and display on the instrument at the designated place.
6.5.12    In case of non-compliance, follow the Maintenance Program.
6.6  Calibration of gradient proportionate valve:
6.6.1        Install union in place of column and flush solvent lines (A & B) at the flow rate of 2.0ml/min with purified water.
6.6.2        Prepare the mobile phase.
6.6.3        Prepare 0.3% acetone with HPLC grade purified water.
6.6.4        Fill reservoir A with 100% HPLC grade purified water and reservoir B with 0.3% acetone in HPLC grade purified water as mobile phase.
6.6.5        Enter the following time set up.
Time
Function
Value
0.01
B conc.
10
10.00
B conc.
10
10.01
B conc.
50
20.00
B conc.
50
20.01
B conc.
90
30.00
B conc.
90
30.01
B conc.
100
40.00
B conc.
100
40.01
B conc.
0
50.00
B conc.
0
6.6.6        Use detector at wavelength of 254nm.
6.6.7         Record the print out of gradient valve test as per Annexure-1.
6.6.8        The gradient valve test shall be accepted if actual concentration with ±1% of set concentration.
6.6.9        Make entry of the column usage in the Column Usage Log register.
6.6.10    Make entry of the usage into the Instrument Usage Log Register.
6.6.11    Compare the result for its compliance against limit given in the Calibration Log and put the remarks regarding the HPLC chromatography Calibration status.
6.6.12    Prepare the calibration status Label and display on the instrument at the designated place.
6.6.13    In case of non-compliance, follow the Maintenance Program.
6.7  Calibration of injector (to check reproducibility and linearity of the injection volume):
6.7.1        Standard  preparation:
6.7.1.1  Take clean and dry 50.0ml volumetric flask.
6.7.1.2  Pipette out 1.0ml of Benzene and Toluene in to the clean and dry 50.0ml volumetric flask.
6.7.1.3  Make up the volume to 50.0ml with Methanol and mix well.
6.7.2        Chromatographic condition:
6.7.2.1  Column: ODS C18, (25 cm x 4.6 mm ID, 5 µm).
6.7.2.2  Mobile Phase: Methanol: Water (70:30).
6.7.2.3  Flow Rate: 1.0 ml/min.
6.7.2.4  Wavelength: 254 nm.
6.7.2.5  Injection Volume: 20 µl.
6.7.3        Calibration:
6.7.3.1  Ensure that, the instrument is ready for calibration and Start-up procedure is followed.
6.7.3.2  Ensure that, the instrument is set according to the Chromatographic conditions.
6.7.3.3  Follow the Instrument Operating procedure i.e. SOP, Inject 10µl in triplicate and record the chromatograms.
6.7.3.4  Repeat the injection of above solution by injecting 15, 20, 25, 30 µl in triplicate.
6.7.3.5  Take the print out of the chromatograms and attach to the Calibration Log.
6.7.3.6  Record the Area and Retention times of the of the Benzene and Toluene peaks in the Calibration Log.
6.7.3.7  Make entry of the column usage in the Column Usage Log register.
6.7.3.8  Make entry of the usage into the Instrument Usage Log Register.
6.7.3.9  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.
6.7.3.10                                                                            Find out the Correlation coefficient “r2” for each peak at five levels and record in the Calibration Log.
6.7.3.11                                                                               Compare the result for its compliance against limit given in the Calibration Log and put the remarks regarding the HPLC chromatography Calibration status.
6.7.3.12                                                                               Prepare the calibration status Label and display on the instrument at the designated place.
6.7.3.13                                                                            In case of non-compliance, follow the Maintenance Program.
6.8  Calibration of detector (D2 lamp energy check):
6.8.1        Ensure that, the instrument is ready for calibration and start-up the procedure as followed.
6.8.2        On the detector’s display window, some values at the functions “l (nm)”, “abs (AU)”, “range (AUFS)” and “lamp” appears.
6.8.3        On the display, previously set value blinks at “l (nm)” functions, enter the wavelength to 254nm by pressing numeric keys.
6.8.4        Press “Func Back” key and select “lamp” functions, enter 1 to select D2 lamp.
6.8.5        Further press “Func Back” key till “REF EN” appears.
6.8.6        Record the reference energy of the D2 lamp at 254nm in the Calibration Log.
6.8.7        Compare the result for its compliance against limit given in the Calibration Log and put the remarks regarding the HPLC chromatography Calibration status.
6.8.8        In case of non-compliance, follow the Maintenance Program.
6.8.9        Standard  preparation:
6.8.9.1  Take clean and dry 50.0ml volumetric flask.
6.8.9.2  Pipette out ml of Benzene and Toluene as per the following table in to the clean and dry 50.0ml volumetric flask, separately.
6.8.9.3  Make up the volume to 50.0ml 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
50.0ml
2
1.0
1.0
50.0ml
3
1.5
1.0
50.0ml



6.8.9.4  Chromatographic conditions:
6.8.9.4.1        Set each of the equipment to the following parameters as per the equipment’s parameter set up procedure:
6.8.9.4.1.1   Column: ODS C-18, (25cm×4.6mm ID, 5m)
6.8.9.4.1.2  Mobile phase: Methanol: Purified water (70:30).
6.8.9.4.1.3  Flow rate: 1.0ml/min.
6.8.9.4.1.4  Wavelength: 254nm.
6.8.9.4.1.5  Injection volume: 20μl.
6.8.9.5  Calibration:
6.8.9.5.1        Ensure that, the instrument is ready for calibration and Start-up procedure is followed.
6.8.9.5.2        Ensure that, the instrument is set according to the Chromatographic conditions.
6.8.9.5.3        Follow the instrument operating procedure, inject each of the solutions in triplicate and record the chromatogram.
6.8.9.5.4        Take the print out of the chromatograms and attach to the Calibration Log.
6.8.9.5.5        Record the Area and retention times of the Benzene and toluene peaks in the Calibration Log.
6.8.9.5.6        Make entry of the column usage in the Column Usage Log register.
6.8.9.5.7        Make entry of the usage into the Instrument Usage Log Register.
6.8.9.5.8        Calculate the area ratio of the benzene to toluene, find out the mean value of the ratios and record in the Calibration Log.
6.8.9.5.9        Find out the correlation co-efficient “r2” from the mean area ratio values of the three levels and record in the Calibration Log.
6.8.9.5.10    Plot the curve for area ratio corresponding to benzene to toluene peaks v/v concentration. Find out the RSD (reproducibility) and record in the Calibration Log.
6.8.9.5.11    Compare the result for its compliance against limit given in the Calibration Log and put the remark regarding the Calibration Status.
6.8.9.5.12    Prepare calibration status label and display on the instrument at the designated place.
6.8.9.5.13    In case of non-compliance, follow the maintenance program.
6.9  Frequency of calibration:
6.9.1        Pump: Once in three months and after each maintenance job.
6.9.2        Gradient: Once in six months and after each maintenance job.
6.9.3        Injector: Once in six months and after each maintenance job.
6.9.4        Detector: Once in three months and after each maintenance job.
6.10                            After calibration status label on the instrument containing “Calibrated on”, “Due on” and “Signature”.
6.11                            Report to QC head, if any discrepancy observed during calibration or operating the instrument and affix ‘Under Maintenance’ label on the instrument.

UNDER MAINTENANCE
Equipment: ___________________________________________
Since:_________________________________________________

Signature: ______________________

7.0  REVISION LOG:
Revision No.
Effective Date
Reason
00

New SOP

8.0  REFERENCES:
8.1  Not Applicable.
9.0  ANNEXURES:
Annexure 1: Calibration of HPLC apparatus.












Annexure: 1
Calibration record of HPLC apparatus
Calibration of HPLC apparatus
Calibration date
Date of last calibration done
Next due for calibration




Instrument details
Instrument name
Instrument make
Instrument identification No.




Observation Chart
1.      Calibration of pressure drop (Leakage test):
Pressure max.
Pressure max. set
Start time
Stop time
Total time
Pressure observed
300 bar






Remarks:




2.      Calibration of flow rate:
Flow rate
(ml/min)
Time required to collect 10.0ml mobile phase
Limit
Theoretical
(in seconds)
Actual with water
Actual with methanol
0.5ml
1200


1194-1206
1.0ml
600


594-606
1.5ml
450


443-457
2.0ml
300


294-306

Remarks:




3.      Calibration of gradient proportionate valve:
PARAMETERS
Mobile phase
Reservoir A 100% Water with HPLC.
Reservoir B 0.3% acetone HPLC.
Wavelength
254nm
Flow rate
2.0ml/min.

Time range
Height from base line to B concentration peak
Value obtained
Actual value set in the program
0.01 to 10.00

Lt: 9.9 to 10.1

10.01 to 20.00

Lt: 49.5 to 50.5

20.01 to 30.00

Lt: 89.1 to 90.9

Value=       Height of B conc.___
   Height of a 100% conc.
Remarks:




4.      Calibration of injector:
a.      Preparation of standard solution:
Taken ___ml (1.0ml) of the _______ benzene and ________toluene into a ______ml (50.0ml) clean, dry volumetric flask, make up the volume with methanol, mixed well.
b.      Chromatographic condition:
Parameter
Test condition
Applied condition
Mobile phase
Methanol: water (70:30)

Column
ODS C-18, (25cm×4.6mm ID, 5μm)

Flow rate
1.0ml/min

Wavelength
254nm


Injection volume
Injection-1
Injection-2
Injection-3
Mean
%RSD (Not more than 2.0%)
Benzene
Toluene
Benzene
Toluene
Benzene
Toluene
10μl
RT









Area








15μl
RT









Area








20μl
RT









Area








25μl
RT









Area








30μl
RT









Area









Co-efficient of co-relations r2: _______________ (Limit: NLT 0.999).

Remarks:




5.      Calibration of detector:
D2-Lamp Energy check:
Wavelength to be set
Wavelength set
Reference energy observed
Limit





a.      Preparation of standard solution:
Solution
(level)
ml of benzene
ml of toluene
in methanol
To be taken
Taken
To be taken
Taken
To be diluted
Diluted to
1
0.5ml

1.0ml

50.0ml

2
0.5ml

1.0ml

50.0ml

3
0.5ml

1.0ml

50.0ml

b.      Mobile phase preparation:
Taken _____ml of ______ (methanol HPLC grade) into a ______ ml clean, dry volumetric flask, added _______ ml of _______ (Water HPLC grade), mixed well, and allowed to cool to room temperature. Filtered through 0.45/0.22μm.
c.       Chromatographic conditions:
Parameter
Test condition
Applied condition
Mobile phase
Methanol: water (70:30)

Column
ODS C-18, (25cm×4.6mm ID, 5μm)

Flow rate
1.0ml/min

Wavelength
254nm

Range
1.0 AUFS

Injection volume
20μl.


Solution
(level)
Benzene area
Toluene area
Area ratio
(benzene/ toluene)
Retention time
Area
Retention time
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

Remarks:















Calibration by/date:
Checked by/date:





10.0    ABBREVIATIONS:
Abbreviation
Expanded Form
SOP
Standard operating procedure
&
And
No.
Number  
Ltd.
Limited
Q.C
Quality control
Q.A
Quality assurance
QCEB
Quality control equipment calibration
F
Format
Sr.#
Serial number
g
Grams
%
Percentage
oC
Degree centigrade
mg
Milligram
SD
Standard deviation
NMT
Not more than


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