This document provides information on calibrating and qualifying various analytical instruments. It discusses the importance of calibration and qualification to ensure proper functioning and accurate results. It describes the different types of qualification including design, installation, operational and performance qualification. It then provides details on specific calibration procedures for various instruments like electronic balances, pH meters, UV-Vis and IR spectrophotometers, and HPLC. The calibration procedures ensure the instruments meet parameters for accuracy, resolution, wavelength verification and flow rate consistency.

1. Calibration and Qualification of Analytical
Instruments
ROHIT BHATIA
ASSISTANT PROFESSOR
DEPT. OF PHARMACEUTICAL CHEMISTRY
ISF COLLEGE OF PHARMACY
WEBSITE: - www.isfcp.org
EMAIL: bhatiarohit5678@gmail.com
ISF College of Pharmacy, Moga
Ghal Kalan,GT Road, Moga- 142001, Punjab, INDIA
Internal Quality Assurance Cell - (IQAC)

2. Flow of Contents
Introduction
Qualification
Types of Qualification
Calibration
Calibration of Electronic Balance
Calibration of PH Meter
Calibration of UV Spectrophotometer
Calibration of IR Spectrophotometer
Calibration of HPLC
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3. Introduction
 The objective of any chemical analytical measurement is to get
consistent, reliable and accurate data.
 Proper functioning and performance of analytical instruments and
computer systems plays a major role in achieving this goal.
 Therefore, analytical instrument qualification (AIQ) and calibration
should be part of any good analytical practice.
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4. Qualification
 It is the Action of proving and documenting that equipment or
ancillary systems are properly installed, work correctly, and actually
lead to the expected results. Qualification is part of validation , but
the individual qualification steps alone do not constitute process
validation.
 Use of a qualified instrument in analysis contributes to confidence in
the validity of generated data.
4

5. Parts of Qualification
1. Design Qualification (DQ)
 Design qualification (DQ) is the documented collection
of activities that define the functional and operational
specifications of the instrument and criteria for selection
of the vendor, based on the intended purpose of the
instrument.
 The Design Qualification activity is most suitably performed by the
instrument developer/manufacturer.
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6. 2. Installation Qualification (IQ)
Installation Qualification is a documented collection of activities needed to install
an instrument in the user’s environment. IQ applies to a new, pre-owned or an
existing onsite—but not previously qualified—instrument. The activities and
documentation associated with IQ are as follows:
 System Description
 Instrument Delivery
 Utilities/Facility/Environment
 Network and data storage
 Assembly and installation
 Installation verification
6

7. 3. Operational Qualification (OQ)
After a successful IQ the instrument is ready for OQ testing. The OQ
phase may consist of these test parameters:
 Fixed Parameters: These tests measure the instrument's non
changing, fixed parameters such as length, height, weight, etc. If the
vendor-supplied specifications for these parameters satisfy the user,
he or she may waive the test requirement. However, if the user wants
to confirm the parameters, testing can be performed at the user’s site.
 Secure Data Storage, Backup, and Archive: When required, secure
data handling, such as storage, backup, and archiving should be
tested at the user site according to written procedures.
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8. Contd…
 Instrument Functions Tests: These test important instrument
functions to verify that the instrument operates as intended by the
manufacturer and required by the user. The user should select
important instrument parameters for testing according to the
instrument's intended use.
8

9. 4. Performance Qualification (PQ)
After the IQ and OQ have been performed, the instrument’s continued suitability for its
intended use is proved through performance qualification. The PQ phase includes
these parameters:
 Performance Checks: PQ tests are usually based on the instrument’s typical on-site
applications. Some tests may resemble those performed during OQ, but the
specifications for their results can be set differently if required. These are carried out
on a running instrument, not on a new instrument.
 Preventive Maintenance and Repair: When PQ test(s) fail to meet specifications,
the instrument requires maintenance or repair. Relevant PQ test(s) should be
repeated after the needed maintenance or repair to ensure that the instrument
remains qualified.
 Standard Operating Procedure for Operation, Calibration, and Maintenance.
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10. Calibration
 The demonstration that a particular instrument or device produces
results within specified limits by comparison with those produced by
a reference or traceable standard over an appropriate range of
measurements.
 The instruments must be routinely calibrated to get the desired
results.
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11. Need for Calibration
 When the instrument is new
 When a specified time has been elapsed
 When operating hours has been elapsed
 When instrument has shock or vibration
 Sudden change in weather
 When observations are questionable
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12. Common Analytical Instruments 12

13. Calibration of Electronic Balance 13

14. Procedure
 Place 500mg standard calibrated weight on the pan.
 Record the display of the weight.
 Remove the weight & again place the weight on the pan & record the display once again.
 Repeat the above operation to get ten readings Calculate the standard deviation of ten
readings.
 The measurement uncertainty shall be considered satisfactory if three times of standard
deviation of not less than 10 replicate weighing divided by amount weight does not exceed
0.001.
 Affix a ‘Calibration label’ on the instrument.
 Report any discrepancy noted at the time of calibration to Q.C Manager & notify the defect
to rectify the instrument. & affix an ‘UNDER MAINTENANCE ‘ label on the instrument.
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15. Drift Check
 For the calculation of the drift 10 mg weight shall be used.
 Note down the 10 measurements of the 10 mg weight in the
performance check .
 Variation in the observed weight from the mean value does
not exceeds ± 0.2 mg.
 The 10 mg weight shall meet the performance check criteria
of the mass value i.e. 0.1% of actual mass value.
 E.g. For all the 10 measurements of 10 mg weight, variation
in the weighing cannot exceed 0.01mg.
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16. Performance Check 16
 After the Auto calibration put 1 mg, 2 mg, 5 mg, 10 mg and 20 mg
weights individually. The measurement shall be within the 0.1% of
actual mass value of the individual weight as given in the performance
check log.
Uncertainty Check
 The measurement of uncertainty shall be carried out by using 10 mg
weight.
 Put the external weight of 10 mg on the pan and note the 10
measurements.
 Calculate the measurement of uncertainty as follows.
 Measurement of uncertainty shall be Not More Than 0.001.

17. Calibration of PH Meter 17

18. Calibration Procedure
Use the squirt bottle to rinse and the plastic beaker to collect the water. Two buffer
calibrations should be done before any measurements are taken. One buffer
calibration should be done every two hours.
1. ALWAYS start with the yellow pH 7 buffers as the first calibration buffer.
2. Use fresh (< 1 week), room temperature buffers. The pH meter does display
temperature, which should be taken into account when using the buffers.
3. The buffers should be stirred using a stir plate and magnetic stirrer while they are
being measured for calibration.
4. Use the two buffers that bracket the expected range of measurements. For example,
if your samples are expected to be acidic, use the pH 7 and pH 4 buffers to
calibrate. If they are expected to be basic use the pH 7 and pH 10 buffers.
5. Rinse the electrode and place into the first buffer.
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19. Contd…..
6. Press the cal keys. CALIBRATION is displayed above the main field and the time
and date of the last calibration are displayed. After a few seconds, P1 is displayed in
lower field. P1 indicates that the meter is ready for the first buffer and a value has not
yet been entered. When READY is displayed, indicating electrode stability, the
reading begins to flash, press the yes key. The display will remain frozen for two
seconds. Then P2 will be displayed in the lower field indicating that the meter is ready
for the second buffer.
7. Rinse the electrode and place into the second buffer. When READY is displayed,
press the yes key. Press the measure key to end calibration. SLP appears in the
lower field with the actual electrode slope, in percent, in the field.
8. The meter automatically advances to the measure mode. MEASURE is displayed
above the main field.
9. Rinse the electrode and place into sample. Record pH directly from the meter
display and temperature from the lower field.
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20. One buffer Calibration
1. Select one buffer that closely approximates the expected sample pH.
2. Repeat steps 5 and 6 from above.
3. Press the measure key. SLP will be displayed in the lower field and
the electrode slope in memory in the main field. If necessary, enter
the correct electrode slope determined by the two point calibration
and press the yes key. If slope value is unknown, enter 100.0 or
perform a two buffer calibration.
4. Rinse the electrodes and place into sample. Read the pH directly from
the main display and temperature from the lower field.
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21. Calibration of UV Spectrophotometer 21

22. Calibration Procedure
Calibration of UV Spectrophotometer is done in four steps:
 Control of wave length
 Control of absorbance
 Limit of stray light
 Resolution power
22

23. Control of Wave Length
 Weigh accurately 1.0 g of Holmium oxide and dissolve it in 1.4 M
Perchloric acid solution. Make up volume up to 25 ml with the same
solvent.
 Select the method file of CONTROL OF WAVE LENGTH in the
instrument.
 After selecting the file press Reference button for base line
correction.
 Then fill the cuvette with 1.4 M Perchloric acid solution and put in
the sample cubicle and press reference to zero.
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24. Contd…
 After auto zero put the Holmium perchlorate solution in the sample
cubicle then press the start key.
 Scan it and verify the absorption maxima of Holmium Perchlorate
solution. The permitted tolerance is given in the following table:
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25. Control of Absorbance
 Dry a quantity of the Potassium dichromate by heating to constant
weight at 130°C.
 Weight accurately about 60 mg of dried potassium dichromate and
dissolve it in 0.005M sulphuric acid solution. Make upto 1000 ml
with the same solvent. Mark the solution as ﴾A﴿.
 Weight accurately about 60 mg of dried potassium dichromate and
dissolve it in 0.005M sulphuric acid solution. Make up to 100 ml
with the same solvent. Mark the solution as ﴾B﴿.
 Select the method file of CONTROL OF ABSORBANCE in the
instrument.
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26. Contd….
 After selecting the file press Reference button for baseline correction.
 Then fill the Cuvette with 0.005M Sulphuric acid for blank and put
in both sample cubicle and press reference to zero.
 After auto zero put the Potassium Dichromate solution labeled as
solution ‘A’ in sample cubicle then press start key taking absorbance
individually for first four wavelength mentioned in ‘Table I’.
 Now take absorbance at 430 nm for solution ‘B’.
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27. Contd…
Note the absorption maxima of Potassium Dichromate solution at
different wave length and calculate the absorbance, tolerance is given in
below table:
27

28. Limit of Stray Light
 Dry a quantity of the Potassium chloride by heating to constant
weight at 130°C.
 Weight accurately 1.20 g of dried potassium chloride and dissolve it
in 50 ml distilled water. Make upto 100 ml with the same solvent.
 Select the method file of LIMIT OF STRAY LIGHT in the
instrument.
 After selecting the file press Reference button for baseline correction.
 Check the absorbance of above solution using water as a blank at 200
nm.
 Absorbance should be greater than 2.0
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29. Resolution Power
 Prepare 0.02%v/v solution of Toluene in Hexane UV.
 Select the method file of RESOLUTION POWER in the instrument.
 After selecting the file press Reference button for baseline correction.
 Measure the absorbance of above solution at 266 nm and 269 nm
using Hexane UV as blank solution.
 The ratio of absorbance maxima at 269 nm to that of 266 nm minima
should be more than 1.5
 Note down the report in the internal calibration certificate and in
Instrument Logbook.
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30. Calibration of IR Spectrophotometer 30

31. Calibration Procedure
 Switch on the system.
 Set instrument parameter as follows.
RESOLUTION 2.0
APODIZATION Strong
RANGE 4000‐400 cm‐1
MODE RATIO
NUMBER OF SCAN 16
 Allow the instrument to warm for 30 minutes
 Operate the instrument as per operation SOP.
 Open the sample compartment cover of FTIR 1600 and place the polystyrene film
in the sample holder and the close the cover.
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32. Contd…
 Click OK in the SCAN MODE.
 The spectrum of the polystyrene film is displays on the screen.
 PRINT the spectrum by going to print function.
 When the printer of the spectra is completely go to DATA and then to
PEAK.
 The peak data’s of the spectra of the polystyrene film is displays on
the screen.
 PRINT out these data’s by going to print function.
 Compare the wave function accuracy with the following limit.
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33. Limit of wave Number Accuracy
3060.0 ﴾±1.5﴿ 2849.5 ﴾±1.5﴿ 1942.9 ﴾±1.5﴿ 1601.2 ﴾±1.0﴿ 1583.0 ﴾±1.0﴿
1154.5 ﴾±1.0﴿ 1026.3 ﴾±1.0﴿
Resolution performance of the apparatus
 Record the spectrum of the polystyrene film 0.04 mm in thickness.
The difference x between the percentage transmittance at the
transmission maximum A at 2870 cm‐1 ﴾3.48 μm﴿ and that at the
transmission minimum B at 2849.5 cm‐1 ﴾3.51μm ﴿ should greater
than 16. The difference y between the percentage transmittance at the
transmission maximum C at 1589 cm‐1 ﴾5.29 μm﴿ and that at the
transmission minimum D at 1583 cm –1 ﴾5.32 μm﴿ should be greater
than 12.
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34. Contd…
 Record the observation in the calibration record Affix a
“CALIBRATION LABEL “on the instrument.
 Report any discrepancy observed during calibration or operation of
the instrument to QA Manager and notify the defect to Engineering.
Dept /Service.
 Affix an ‘UNDER MAINTENANCE; label on the instrument.
Frequency of Calibration
 Once in three month and after each maintenance job.
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35. Calibration of HPLC 35

36. Calibration Procedure
1. Calibration of Pump
 Set the flow rate to 1.0 ml/min.
 Connect Resisto tubing 2 meter x 0.1 mm ID. in place of column.
 Start the pump and wait till equipment stabilised or a baseline is achieved.
 Weigh the dry empty A grade measuring cylinder. Weigh & record the same.
 Collect the water from outlet of Resisto tubing for 5 minutes and weigh the
measuring cylinder. Record the value.
 Repeat the previous step and continue the process for four repetitions.
 Calculate the flow rate accuracy.
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37. 2. Wavelength Accuracy of the Detector 37
Switch on the LC 2010 C, LC 2010 C HT by pressing function key.
Press VP key.
Press Func Key and then Press F5.
Press 5 for detector.
Press Right Arrow Key, to go to next screen (2/5).
Press Down Arrow Key seven times (make sure the cursor is blinking in ‘z wave’
parameter) press 1 enter.
Press Func and Press F5 for exit.
Press Met Key.
Flush the flow cell with Methanol.
Open the port of Detector and pass the methanol with using the syringe for cleaning.
Press Zero Key and ensure that the absorbance is zero.
Inject 5 ml of 2 mg/100 ml solution of caffeine in methanol and changing the
wavelength from 266 nm to 277 nm with the step of 1nm and record the respective
absorbance.
Record the wavelength that produces the maximum absorbance.
Record the results.

38. 3. Reproducibility of Results
 Prepare the mobile phase consisting of 70 % Methanol: 30 % Water.
 Prepare a mixture of 0.1 % v/v Benzene and 0.1 % v/v Toluene in Methanol.
 Set the flow rate of 1 ml/min.
 Allow the system to be saturated with the mobile phase for at least 15
minutes before injecting the sample preparation.
 Record the area and Relative Retention time of both benzene and toluene.
 Calculate the RSD for both area and Relative Retention time respectively.
 RSD of Area should not be more than 2.0 %. & RRT of Benzene & Toluene
should not be more than 2.0 %.
 Calculating the Tailing Factor ( Asymmetry), Resolution and Theoretical
Plate and record.
38

39. 4. Linearity Test
 Prepare the mobile phase consisting of 70% Methanol: 30 % water.
 Stock solution preparation: Prepare a mixture of 0.2 % Benzene and
0.2 % Toluene in methanol.
 Make dilution in following manner.
 Take 5 ml, 10 ml, 15 ml, 20 ml of stock solution respectively in 25 ml
of volumetric solution and make up with methanol.
 Set flow rate at 1.0 ml/min.
 Allow the system to be saturated with mobile phase for at least 15
minutes before injecting the different solutions.
 Record the area of both benzene and toluene.
 Calculate the Linearity for both Benzene and Toluene respectively.
39

40. 5. Carry Over Test
 After completion of the Linearity test, inject 20μl of methanol.
 Calculate the Carry Over test.
40

41. 6. Temperature Accuracy Test For Column Oven & Sample Cooler Tray
 Use a calibrated thermometers to measure the actual temperature in the oven
& of sample cooler tray and compare it to set value
 Procedure for Temperature Accuracy test for Column Oven.
 Set the temperature accuracy is measured by using “ Performance Check ”
 Press [VP] [F3 (VALID)] [2] [Enter] key, and select “ 5 oven Temperature”
from the “ Performance Check ” menu.
 Select the 40°C and 50°C temperature to be measured as instructed on the
screen.
 Use the clip to set a calibrated temperature sensor at the center of the lower
step of heat block inside the device .
 Press the [OVEN] key to start temperature adjustment. After the temperature
has stabilized the temperature fluctuation amplitude will be automatically
measured for 5 minutes.
41

42. Contd…
 Record the Actual value against the calibrated thermometer value for
different time intervals of 30, 45 and 60 minutes.
 Procedure for Temperature Accuracy test for sample cooler tray
 Press the [Seq], [F1] key, and enter “4” to “cooler temp” ( auto temperature
setting parameter).
 Enter 1 to “ SMPL.C” in other parameter section of Method screen.
 Set the cooling rack ( R ) in the main unit & put the probe of calibrated
thermometer inside the sample cooler tray.
 Wait to stable at the set temperature since it normally takes around 90 min.
 Press [VP] F3 (VALID)] [2] [Enter] key, and select “ 11. Cooler
Temperature” from the “ Performance Check”.
 Record the Actual value against the calibrated thermometer value for
different time intervals of 30, 45 and 60 minutes.
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43. 43