Techniques protocols for Instrumental Analysis ( calibration and Instrumentation of HPLC,UV-Spectrophotometer,PH meter etc)

1. Techniques Protocols for
Instrumental Analysis
PRESENTED BY:AREEBA SHAFIQ
M.PHILL PHARMACEUTICS SCHOLAR (ISLAMIA UNIVERSITY
BAHAWALPUR,PAKISTAN.

2. Contents
HPLC
UV-SPECTROPHOTOMETER
PH METER
DIGITAL STIRRER
CENTRIFUGATION Machine
Viscometer
Weighing Balance

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

4. Purpose of Equipment Calibration
• To determine the Precision and Accuracy of the parameters of an equipment in
relation to its use.
• To determine the Uncertainty of the Parameters within a stated confidence level
(usually 95%).
• To maintain traceability of the results provided by the equipment.

5. Parameters of an Instrument to be
Calibrated
 Any Parameter of an instrument that will affect the quality of the products/test
results has to be calibrated
 (e.g.,a refrigerated centrifuge :speed,time,temperature)

6. General Requirments of Equipment
Calibration
 All Equipment must be uniquely identified and Labelled.
 Acceptance criteria should be set for each type of equipment.
 ALL equipment should be calibrated prior to being placed into service,recalibrated on
schedule and after major repair.
 Equipment Calibration method must meet the accuracy and precision requirements of
the equipment.
 External Equipment Calibration shall be performed by Organizations nationally
accredited for the Equipment Calibration.
 In-house Equipment Calibration must be performed by trained and Competent staff
using reference standards traceable to national standards.

7.  Equipment Calibration records should include the acceptance ranges and results
sufficient to support the accuracy and Precision of the equipment within the
acceptance ranges.
 All equipment shall carry calibration status labels with at least the following
information:
• Equipment Identification
• Date of Calibration due
• Signature of Authorized Staff

8. Equipment Calibration Label

9. Calibration Method
 Determine the accuracy of a Parameter of an instrument by comparing with a
reference value.
 Determine the precision of a parameter of an instrument by repeat measurments.

10. Requirments of Reference equipment
used for Calibration
 The reference equipment must have a certified accuracy traceable to
international/National Standards and at least three times(preferably five times)
better than that required for equipment being calibrated.
 The reference equipment should only be used for calibration purpose and stored
securely.

11. Intervals for Equipment Calibration
 It depends on the frequency of the equipment being used,its condition and
previous calibration results.
 It should ensure detection of malfunction and continued optimal function of the
equipment.
 It should be determined by each institute for each equipment in use based on the
manufacturer’s instruction or national guidelines.
 It varies from one month to ten years.

12. Weighing Balance
A weighing balance is an instrument that is used to determine the weight or mass of an object.

13. Calibration of Balance
Requirments:
 Every three years:full calibration comprising accuracy,precision,off-center loading
and hysteresis check
 Every six months:Precision
 Daily or day of use:scale value check
 Each weighing :zero check

14. Calibration of Balance
Off-center Loading test
• This test is to determine the effect of eccentric loading of the balance
• Use a nominal mass of approximately 1/3-1/2 of the full range of the balance
• Weigh the mass successively at the center on the front,rear ,left and right position
on the lip of the pan by the symmetrical weighing method.(the ‘lip’of the pan is
the area where the flat surface of the pan starts to curve up)

15. Calibration of Balance

16. Calibration of Balance
Precision Check
• Take a minimum of 10 successive readings at near zero weight,half range and full
range of the balance .
(Note :the balance should not be zeroed throughout the successive weighting)
• Calculate the σ for these three series of readings
• Take the largest σ as the σ of the balance
• The σ should not be greater than the 0.0005 gm for 200 gmfour-digit balance.

17. Accuracy Check( 5~10 Point check)
Weigh a single reference mass of value ‘M’ (1/10th of the full range of the balance )
Calculate the correction of each check point (Ci=M-(mi-zi))
Calculate the limit of Performance of the balance F=3 σ + max.of Ci

19. Hysteresis test
This test checks the variability of a measured mass when its value is reached from the
possible directions of the scale (i.e direction of increasing load and direction of decreasing
load)
Use a Nominal mass,M,~1/2 of the full range of the balance
Zero the balance, Z1

20. Hysteresis test
Place the mass on the pan and record the scale reading m1
Add extra weights to the Pan until the balance reads full –range.
Remove extra weights (Do not remove the mass) and record scale reading m2
Remove the mass and record zero z2
Repeat the test three times and express the hysteresis as the average of the
differences between (m1- m2) and (z1 - z2).

22. Daily Scale check
• Check the balance with a mass close to the maximum capacity
the balance or to the weight to be usually weighed.
• It should be within ±3σ.
• Check the mass every six months and re- state the weight of the
mass if it is out of the range.

23. Acceptance Criteria
 Off-centre loading: not more than σ.
 Hysteresis : not more thanσ.
 F (3σ + max. of Ci): not higher than the resolution of balance (e.g.,
0.0005 gm for a 200 gm 4-digit balance)
 Daily Scale check: within 3σ.

24. Operation of Weighing Balance
 Check that all connections of the balance are proper & balance is On.
 Ensure that balance is placed on shockproof bench top .
 If the balance is showing some reading on the display then(ensure that nobody
requires that reading press tare key.
 Balance should show 0.000
 Open the door from one side & keep container or sampling bag as required
&close the door & press tare key.
 Then open the door & put required sample.
 Add sample till you get desired weight on display & close the door .
 After getting a stable display ,open the door & take out the sample with
container or sampling bag
 Close the door & press tare key again. (2)

25. Weighing balance

26. Centrifuge
A centrifuge is a device that uses centrifugal force to separate various components
of a fluid. This is achieved by spinning the fluid at high speed within a container,
thereby separating fluids of different densities (e.g. cream from milk) or liquids from
solids.

27. 2. Calibration of Centrifugation Machine
Requirements:
 Speed :
Quarterly by Tachometer
 Timer :
Quarterly by calibrated timer
 Temperature :
Semi-annually by calibrated thermometer

28. Centrifugation machine

29. Acceptance Criteria:
 Speed :
±10% of the set or displayed speed
 Timer :
±10% of the set time
 Temperature :
±2oC

30. Viscometer
Viscosity is measure of fluid’s resistance to flow. It is to drive a spindle ( which is
immersed in the test fluid ) through a calibrated spring. The viscous drag of the fluid
against the spindle is measured by the spring deflection. Spring deflection is
measured with a rotary transducer.

31. Calibration of Viscometer
Procedure of Brookfield Viscometer Calibration
• Ensure that all the connections of Instrument are Proper.
• Turn the Viscometer on
• Takeout the viscosity Standard Solutions in 500 ml beaker,adjust the temperature
to 25ͦC. Attach the required spindle ,adjust require speed %measure the
viscosity.,note down the constant reading.
• Viscosity standards,speeds,spindles required&tolerance limit are as follows:

33. Acceptance Criteria
• Viscosity should be within the tolerance limit
• Do not transfer the viscosity solution to original container back after use.
Precautions
Take care to avoid any contamination of the standard solutions.
Avoid air bubble entrapment during transfer of Standard solutions.
Frequency
Once in 6 months

34. Brookfield Viscometer

36. Operation of hplc
• Check the general cleaning of instrument.
• Switch on the instrument allow displaying the ‘Menu Screen’.
• Keep the mobile phase bottle after filteration and degas.
• Purge the mobile phase line by pressing the ‘PURGE’ Button provided below to
the display on the instrument.
• Connect the HPLC Column whichever required ,as per flow direction mentioned in
column.
• Set the flow rate .Column oven temperature and initial wavelength on system
display.

37. References
1. https://jbsr.pharmainfo.in/documents/vol2issue2/2010020204.pdf.
2. https://www.pharmaguideline.com/2010/08/normal-0-false-false-
false_13.html#:~:text=Open%20the%20door%20from%20one,with%20container%
20or%20sampling%20bag.
3. http://www.pharmatips.in/Articles/Pharmaceutical-Equipment/Brookfield-
Viscometer-Operation-
Calibration.aspx#:~:text=It%20is%20to%20drive%20a,measured%20with%20a%20
rotary%20transducer.
4. https://www.slideshare.net/solairajananant/calibration-and-validation-of-
analytical-instruments

38.  http://www.pharmaguidesline.com/sop-for-operation-and-calibration-of-hplc/
 https://www.omega.com/en-us/resources/ph-
meter#:~:text=pH%20meter%20is%20an%20instrument,scale%20of%200%20to%
2014.
 https://www.pharmaguideline.com/2010/01/sop-for-operation-and-calibration-
of-ph.html