calibration of instruments, validation.

1. Instrument
Calibration
Shaikh Asif Jamil
M pharmacy (Pharmaceutics)
Jamia Hamdard

2. Definition of Calibration: ICH
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.
Or
calibration is the comparison of measurement values delivered by a device
under test with those of a calibration standard of known accuracy

3. Importance of Calibration
Accurate reading
Readings are consistent
Reliability of readings
Calibration is required under some conditions:
Specified time period
Instrument is purchased
Specified usage
Sudden climatic change
Errors

4. Calibration of UV-Visible Spectrophotometer
Control of Absorbance: Pot. Dichromate. Dry it completely, weight it 57-63mg add in vol.
flask. Make up volume to 1000ml with 0.005M H2SO4. Take absorbance at 235, 257, 313,
350. Calculate A (1%, 1cm) at each Wavelength.
A(1%, 1cm) =Abs x 10000/wt of pot. dichromate (mg)
Acceptance criteria:
Wavelengths A [1% 1cm] (Limit)
235 124.5 122.9 - 126.2
257 144.0 142.8 - 145.7
313 48.6 47.0 - 50.3
350 106.6 105.6 - 108.2

5. Preparation of reagents :
Sulphuric acid 0.005M: Transfer about 100 ml distilled water into 1000ml
Volumetric flask. To this add 0.27ml of concentrated sulphuric acid. Mix the content
of flask to form homogenous mixture. Make up the volume of the flask using d.w.
Potassium dichromate solution in 0.005M Sulphuric acid
Transfer about 60mg (57-63mg) previously dried Potassium dichromate into 100ml
of Volumetric flask. To this add about 10ml of 0.005M H2SO4. Shake the contents of
flask to affect the solution. Make up the volume upto 100ml with 0.005M H2SO4.
Transfer 10ml of this solution into another 100ml volumetric flask and make up the
volume using D.W.

6. Limit of stray light:
Stray light in any undesired light reaching detector other than selected.
Prepare 1.2%w/v sol of KCl. Weigh accurately 12 gms of KCl in 100 ml
volumetric flask. To this add 20ml DW and shake the contents of the flask to
affect the solution. Make up the volume upto 100 ml with DW.
Measure the absorbance at 198, 199, 200, 201, 202 nm.
Abs should be greater than 2.

7. Resolution power: Record spectrum of 0.02% v/v sol of toluene in hexane in
the range of 260-420nm. Calculate the ratio of absorbances to maxima and
minima.
Acceptance criteria: The ratio of abs at absorbance maxima of 269nm to
that at the absorbance minimum 266nm should not be less than 1.5

8. Calibration of FTIR
Switch on the machine and stand it till IR gets stabilize.
Take Polystyrene film in the sample holder and run it as calibration standard.
Compare spectra with the standard polystyrene spectra.
All the peaks should lie on the test spectra.
Compare height of the peaks.
The height of the observed peak should not be less than 95% of the
standard peak.

9. Compare the values of the obtained peaks with those of the standard values given
in IP for polystyrene.
The spectrum appears with the wavenumbers and %T on the display
Comparer the data with standard IR spectrum of polystyrene film present in
Indian pharmacopoeia.
Compare the data with the following limits, 3060.0 (± 1.0), 2849.5 (± 1.0), 1942.9
(± 1.0), 1601.2 (± 1.0), 1583.0 (± 1.0), 1154.5 (± 1.0), 1026.3 (± 1.0)

12. Electronic Balance

13. Accuracy:
✓Place a standard weight for ex. 5gm on the weighing platform. Record the
weight displayed on the display panel.
✓Determine the difference between the labeled weight and the displayed
weight
✓Repeat the same procedure with increasing weights.

14. Precision:
✓ Place a standard weight for ex. 5gm on the weighing platform.
✓ Record the weight displayed on the display panel
✓Repeat the procedure for 9 times and determine the standard deviation
o Corner load test
✓A standard weight is placed first on the centre of the platform and then on
the four corners of the platform
✓The weight displayed at all positions must be same

15. Calibration of HPLC
LEAKAGE TEST (BY PRESSURE DROP)
1. Switch on the instrument.
2. Place inlet tubing of the Pump in to the Water HPLC grade through suction filter.
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. 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.

16. FLOW RATE CALIBRATION
1. Switch on the Instrument.
2. 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.
3. Wait till the collected mobile phase reaches 10 ml mark of the volumetric flask and Stop
the stopwatch.
4. Record the time required to collect the 10 ml mobile phase in calibration log.
5. Repeat the procedure for 1.0 ml, 1.5 ml and 2.0 ml/ min. flow rates.
6. Repeat the step but using methanol HPLC grade as mobile phase instead of water.
7. The acceptance criteria for Flow Rate accuracy is ± 0.5% of the set flow rate.

17. Injector Accuracy:
1. Connect the pump and detector inlet with union.
2. Prepare mobile phase consisting of a mixture of water and Methanol (70:30 v/v).
3. Set a flow rate of 0.5 ml/m and a run time of 1 m.
4. Set the column temperature at 25± 2°C.
5. Fill a standard HPLC vial to 2/3rd with Milli-Q water. Seal the vial properly with
a cap.
6. Weigh the vial and record the weight as W1 grams.
7. Place the vial in the chromatographic system and perform 6 injections of 50µl
volume from this vial.
8. Weigh the vial again and note the weight after the injections as W2 grams.
Calculate the mean volume injected per injection as follows: Mean injected
volume (µl) = (W1 – W2) ×100/6
9. Acceptance criteria: The mean injected volume should be 50.0±1.0 µl.

20. 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.
Chromatographic Condition :
Mobile Phase : Methanol : Water (70 : 30)
Flow Rate : 1.0 ml/min.
Wavelength : 254 nm
Injection Volume : 20 μl
ml of Benzene to be taken ml of Toluene to be taken To be Diluted with Methanol to
0.5 1.0 50 ml
1.0 1.0 50 ml
1.5 1.0 50 ml

21. Calibration :
1. Ensure that, the instrument is set according to the Chromatographic conditions.
2. Follow the Instrument Operating procedure, Inject each of the Solutions in triplicate and
record the chromatograms.
3. Take the print out of the chromatograms and attach to the Calibration Log.
4. Record the Area and Retention times of the of the Benzene and Toluene peaks in the
Calibration Log.
5. Calculate the Area Ratio of Benzene to Toluene, Find out the Mean value of the ratios
and record in the Calibration Log.
6. Find out the Correlation coefficient “r2” from the Mean area ratio values of the three
levels and record in the Calibration Log.
7. 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.

22. Gas Chromatography
Calibration of Carrier Gas Flow setting-
Touch on “tool” at right hand corner of screen on instrument by using stick provided with
instrument.
Select option utilities.
Touch on column flow calc.
Enter column length, diameter, flow and column oven temperature; it will display required
pressure to set by using carrier pressure knob.
Column oven temperature calibration:
Insert the calibrated digital thermometer from topside by piercing glass wool.
Set column oven temperature on GC to 50°C, 100°C, 200°C, and 250°C.
Allow to stabilize the set temperature for 5 min and record temperature on digital
thermometer at respective temperatures.
Frequency - Once in three months.

23. Detector Calibration:
Prepare 1.0% v/v toluene solution in n-Hexane
Set the chromatographic conditions as follows
Injector temperature : 180°C
Detector temperature : 220°C
Column flow : 2.7 ml/min
Oven temperature : 110°C
Run Time : 5 minutes
Inject blank and then six injections of 1.0% toluene solution in system and record
chromatogram for all injections
Calculate the RSD for the area of six 1.0 % toluene injections.
The RSD should not be more than 15.0%

24. Calibration of Photoflurometer