IT INCLUDE HPLC, UV VISIBLE SPECTROSCOPY, IR AND ELECTROM=NIC BALANCE CALIBRATION IN DETAILS.

1. CALIBRATIONS
BY- ASST. PROF. SANJAYKUMAR UCHIBAGLE,
GHRU, SAIKHEDA.

2. Calibration of Analytical Instruments-
UV-VIS Spectrophotometer and
HPLC

3. What is Calibration?
Calibration is an operation which is performed to assure that the
instrument readings are accurate with reference to the
established standards.
The aim of the calibration program is to ensure that all measuring
and testing equipment included in the program are calibrated
within the manufacturers accuracy specifications or the tolerance
required for the application.

5. Calibration of UV-VIS spectrophotometer
involves
➢Calibration for wavelength accuracy.
➢Calibration for absorbance measurement.
➢Gratings performance or stray light test.
➢Resolution power.

6. Calibration for Wavelength Accuracy:
➢ Take two empty cuvettes, into one cuvette add two
drops of benzene and drain it.
➢ Place the cuvette in sample cell holder with benzene
vapours occupied in it.
➢ Place the empty cuvette in reference cell holder.
➢ Scan it in between 240-270nm.
➢ The maximum absorbance should be at 253.9 ± 0.51 nm.
➢ Absorbance should be below 1 (one).

7. Calibration for Absorbance Measurement:
➢ Prepare 60ppm of potassium dichromate mol wt 294.185
g/mol by using 0.01M sulfuric acid as solvent.
➢ Place the solution in sample cell holder.
➢ Place blank 0.01M sulfuric acid in reference cell holder.
➢ Measure the absorbance values at different λ max values.

8. Gratings Performance or Stray Light Test:
➢ Prepare 1.2% potassium chloride solution in
distilled water.
➢ Distilled water is taken as reference.
➢ Place the potassium chloride solution in sample
cell holder.
➢Absorbance is measured at 195-220nm.
➢Absorbance is must be greater than 2 at 198nm.

9. Resolution power:
➢When prescribed in a monograph, record the
spectrum of a 0.02% v/v solution of toluene in
Hexane.
➢ The ratio of the maximum absorbance at about
269 nm to that at the minimum absorbance at
about 266 nm should NLT 1.5 unless otherwise
specified in the monograph.

11. ANY QUE?

12. Thank You!

13. Calibration of HPLC involves
following parameters:
➢Calibration of Pump
➢Calibration of Injector
➢System Precision
➢Calibration of Detector
Calibration of HPLC

15. Calibration of HPLC involves
following parameters:
➢Calibration of Pump
➢Calibration of Injector
➢System Precision
➢Calibration of Detector
Calibration of HPLC

16. Calibration of Pump
Flow Rate Accuracy:
➢ Prime all the solvent lines with HPLC grade water.
➢ Set the flow rate to 0.5 ml/min.
➢ Wait for about 15 min to stabilize the system and ensure that the pressure is
stable.
➢ Insert the outlet tubing into a 10 ml V.F. and start the stop watch simultaneously.
➢ Stop the stopwatch when the lower meniscus reaches the 10 ml mark V.F. .
➢ Record the elapsed time.
➢ Similarly check the flow for 1.0 ml/m and 2.0 ml/m.
➢ The time taken to collect the water should be with in ± 2.0% of the actual value.

17. Calibration of Injector
Injector linearity
➢ Duplicate injections of 10μg/ml caffeine solution
are injected by setting 5μl, 10μl, 15μl and 20μl
respectively one after one as injection volumes.
➢ The mean peak areas for the above injected
volumes are noted.
➢ Calculate the correlation coefficient by using
linearity curve and the value should be with in the
 acceptance criteria i.e., NLT 0.99.

18. Calibration of Injector
Injection volume Accuracy
➢ HPLC vial is filled with water (HPLC grade) and weighed, it’s
weight is recorded as W1
➢ The vial is placed in the injection tray, then inject six times.
➢ After completion of six injections remove the vial and weigh, it’s
weight is recorded as W2
➢ The injection volume is calculated using the formula,
Acceptance criteria: The mean injected should be 50.0±1.0 μl.

19. System Precision
➢ Standard Preparation: Accurately weigh and transfer
about 60mg of Caffeine into a 100ml volumetric flask.
Dissolve and dilute to the volume with mobile phase.
Transfer 10ml of this solution into a 100ml volumetric flask
and dilute to the volume with mobile phase.
➢ Procedure: Inject blank, followed by standard preparation
in 6 replicates. Note down the areas and retention times. Now
calculate the %RSD of retention time and peak areas for 6
replicates injections.
➢ Acceptance criteria: The %RSD of retention time &
peak area should be <1.0%.

20. Calibration of Detector
Detector Linearity
➢ Prepare a standard stock solution of caffeine(1000μg/ml).
➢ From the stock solution prepare solutions of Concentration
1, 5, 10, 50, 100 μg/ml respectively.
➢ Duplicates of each concentration are injected.
➢ The mean peak areas of all the concentrations are recorded.
➢ Calculate the correlation coefficient value by using linearity
curve and it should be within the acceptance criteria .i.e.,
0.99.

22. Calibration of Detector
Wavelength Accuracy
➢ The wavelength of detector is adjusted to 266nm.
➢ Then inject duplicates of 10μg/ml solution of caffeine and
then the peak area response is recorded.
➢ The wavelength was increased successively at an increment
of +1nm (267, 268, 269……, 276nm) and the peak area is
recorded at all the wavelengths.
➢ The maximum peak area response has to be obtained at
273±2nm (acceptance criteria).

24. ANY QUE?

25. Thank You!

26. Calibration Gas
Chromatography.

31. Calibration of Balance

32. Calibration. Calibration procedures shall include specific directions and limits for
accuracy and precision. When accuracy and precision limits are not met, there shall be
provisions for remedial action to re-establish the limits and to evaluate whether there was
any adverse effect on the device's quality. These activities shall be documented.
Procedure:
•Check the cleanliness of the area.
•Check that platform and exposed parts of the balance are clean and dry.
•Check the level of the balance with the help of spirit level. Adjust the level,
if not levelled.
•Switch on the main power supply of the balance.
•No repairs should be made to any balance by anyone other than a qualified
validation person.
•Do not use balance if it is not calibrated.

33. •Following parameters to be checked while performing
calibration.
1.Accuracy
2.Linearity
3.Precision
4.Corner Load Test
•Frequency: Every Month (± 7 days)
5.1 Accuracy
Check the accuracy of the balance by using 5 standard stamped weights.
Place standard weight one by one in the increasing order in the center of the platform
and record the observations in the balance calibration record.
Acceptance Criteria: Standard Weight ± 2 x Least Count
5.2 Linearity
Draw the linearity curve for the above readings and find out the correlation factor.
Record the observations in the balance calibration record.
Acceptance Criteria: Limit: Not Less Than 0.9999

34. 5.3 Precision
Check the Precision of the balance by using standard weight equivalent to 5 % of
Maximum capacity.
Repeat the procedure for five times and record the readings.
Repeat this using standard weight equivalent to 50 % of maximum capacity.
Calculate Relative Standard Deviation (RSD) for both the standard weights. Record
the observations in the calibration log.
Acceptance Criteria: % RSD Not More Than 0.5 %
5.4 Corner Load Test
Place standard weight equivalent to 30 % of maximum capacity in four corners and
centre of the balance and note down the readings in record. Calculate % Relative
Standard Deviation (RSD).
Record the observations in the balance calibration record.
Acceptance Criteria:
Deviation: Standard Weight ± 2 x Least Count
% RSD: Not More Than 0.5 %

37. Types of balance Analytical balances are designed to measure small masses from
around 320g to sub-milligram. They are very sensitive pieces of equipment so
need to be treated with care. The main types of laboratory balance are (masses
stated are general values only):
• Top-pan balance (200g – 0.001g)
• Analytical balance (320g – 0.0001g)
• Microbalance (6g – 0.000001g)
• Ultra microbalance (6g – 0.0000001g)

38. Trouble shooting The accuracy and precision of an analytical balance must be
guarded and checked at regular intervals. There are many factors that govern
whether an analytical balance behaves itself:
• Gravitational acceleration differences across the globe mean that the balances
calibration may require local adjustment.
• Temperature: Balances take time to equilibrate to laboratory temperature
changes. Also, hot or cold objects can create convection currents in the air which
can cause variation in mass measured. For these reasons it is important to let the
balance and objects equilibrate to the same temperature.
• Moisture: Objects or materials that absorb moisture can appear to gain weight.
This may particularly be an issue for objects that have recently been removed from
a desiccator. Other materials may evaporate or sublime during measurement
Air flows: Air movement in the laboratory across the pan will cause variations in the
measurement. A draft shield reduces this but it will take time for the air within the
draft shield to stabilize once the door is closed. Changes in air temperature within the
draft shield will also cause air movement. These changes can be due to the
temperature of the mass, hands, etc. Reducing air flow incident on the balance in
the laboratory is key to reducing this issue and ensuring all items entering the
draft shield are equilibrated to ambient temperature – using tweezers, not your
warm hands, to move items can help.

39. Static electricity: This can be one of the biggest causes of frustration when using a
balance. If the mass you are measuring wonders up or down and will not stabilize
then there is a good chance that you have a static issue. The static-electrical field
interferes with the electromagnetic field of the balance. To prevent this you can use
an anti-static device which will “fire” positive and negative ions at the weight boat,
powder, etc. to neutralize the static charge. The good anti-static system are incredibly
effective and can save hours of pain and frustration. Anti-static plastic weigh boats
or metal weigh weight boats can also help.

40. Drift in Measurements with Analytical Balances
 Pharmaceutical laboratories and bioscience research institutes make extensive use
of analytical balances that are highly sensitive.
 These analytical balances are greatly affected by their environment and also by
the way they are installed and handled.
 This is why it is important to assess the lab environment to make the required on-
site adjustments.
 The weighing equipment you use in a lab should always deliver accurate results
and all the elements that can cause any discrepancies should be eliminated.
 The opening and closing of a freezer door can cause the temperature to
fluctuate and this should not occur for optimal conduction of weight
measurements.
 Analytical balances are designed to deliver extremely precise results — as low as 1
millionth of a gram and hence they are in use for quality checks in the
production processes of many pharmaceutical manufacturing companies.a

41. What Causes Drift in Analytical Balances & How to Avoid It
 The phenomenon of drift can occur adversely affecting analytical
balances when weighing compounds. It refers to unstable weight
readings, which typically occur due to static charge and
inconsistent temperatures.
 Drift can cause changes in the measurements and leads to
imbalances in displays. If enough static electricity is present in
the environment, readings also become unstable.
 Even if not applying any weight. Pharmaceutical production
lines areas are generally kept clean under highly controlled
conditions.
 The humidity levels are generally below 20% with 24- hour air
conditioning.
 This creates a dry environment and hence any movement of
objects can cause friction. The friction can create static
electricity which can lead to considerable errors and
discrepancies in weighing measurements ranging up to dozens
of milligrams.

42. Ways to Avoid Analytical Balance Drift
 In order to keep static electricity from building up and causing the
drift effect, it is essential to ensure that humidity levels for
weighing equipment are raised to 40% at the time of installation.
 Despite that, if the static energy keeps accumulating and the rate
of electrical discharge is slow, weighing operations should not be
made until the electrical charges are eliminated from the weighing
sample.
 After weighing samples should not be stored in plastic containers
as they are porous and operators should always conduct weighing
operations while standing on anti-static flooring.
 Another external factor that has a dramatic impact on the
accuracy and stability of analytical balances is temperature.
Temperature control is crucial in avoiding the drift phenomenon.
 This includes maintaining constant temperatures in the
environment as well as for your weighing equipment. The best way
to ensure temperature stability is to maintain a variation of not
more than two degrees round the clock. Also the weighing
instrument should remain on at all times so that the temperature
remains consistent.

43. Evaluating the Performance of Analytical Balances
You need to evaluate analytical balances regularly to determine whether they need any
on-site modifications, repairs, or calibration. Problems can occur due to defective
components or because of how users operate them. You should test them for
repeatability to ensure that they are delivering accurate results consistently for a given
object.
• The best test of repeatability is to use a solid, non-magnetic and nonporous container.
Or use a test weight and weigh it repeatedly after returning to zero at the end of every
weighing cycle. Also you can try weighing two objects separately that are exactly half of
the total weighing capacity. Then the difference between the two readings should be less
than the actual tolerance for accuracy.
• If numerical readings turn blank or become frozen, it is safe to assume that your
equipment has a contaminate, is damaged, or mishandled and thus is not producing the
desired results.
• Cornerload is term that indicates the ability of an instrument to generate the same
readings for an object, regardless of where it is placed on the weighing pan. At all the
positions on the weighing pan, the readings should be the same. And if there are errors,
they can be fixed during a field service.
• Linearity testing is done to ensure that the weighing instrument is delivering the same
sensitivity throughout its functional range.