Calibration of Instruments

• ICH definition : 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 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.
• Calibration may be called for:
• A new instrument.
• After an instrument has been repaired or modified.
• When a specified time period has elapsed.
• Before and/or after a critical measurement.
• After an event, for example
• After an instrument has had a shock, vibration, or has been
exposed to an adverse condition which potentially may have
put it out of calibration or damage it.
• Sudden changes in weather.
• Whenever observations appear questionable or instrument
indications do not match the output of surrogate instruments.
As specified by a requirement, e.g., customer specification,
instrument manufacturer recommendation
Advantages of Calibration
• greater assurance of quality
• Quality Control
• Reduces Chances of Error
• Cost Effective Auditing
• Increase Consistency and Reliability
Prevent and Predict Process Failures
 Electronic balance
• USP General Chapter 41 "Balances" is mandatory and
states the requirements for balances used for materials
that must be accurately weighed. Weighing should be
performed using a balance that is calibrated over the
operating range and meets the requirements defined for
repeatability and accuracy.
• USP General Chapter 1251 "Weighing on an Analytical
Balance" is a guideline applicable to balances used in all
analytical procedures.
• It provides information on installation and operational
qualification (IQ/OQ), performance qualification and
balance checks.
• Weighing is a frequent step in analytical
procedures, and the balance is an essential
piece of laboratory equipment in most
analyses.
• In spite of this, weighing is a common
source
of error that can be difficult to detect in
the final analytical results
• The weighing procedure can be separated into
three basic steps:
❑Planning
❑checking the balance and weighing the material.
 Balance Uncertainties
• Check the sample, the balance, and the laboratory
environment for the following causes of errors, and
eliminate them:
• 1. A balance door is open.
• 2. Temperatures of the balance and the material to be
weighed are not the same.
• 3. The sample is losing or gaining weight.
• 4. The balance has been recently moved but has not
been allowed to equilibrate to its surroundings or has
not been recalibrated.
• 5. Air currents are present in the laboratory.
• 6. Temperatures in the laboratory vary.
• 7. The balance is not properly leveled.
• 8. Laboratory operations are causing vibration..
• Typically, the weighing of a sample or standard is the
first step in the analytical procedure, followed by
dilution and subsequent analysis by techniques such
as HPLC or qNMR.
• Any error in the weighing step has the
potential to propagate through the whole
analytical process, causing inaccuracy in the
final result.
• To avoid this situation, the United States
Pharmacopeia (USP) has set stringent requirements
for balances that are used to weigh analytes for
quantitative assessments. These requirements are
designed to ensure that any uncertainty in weighing
is small or even negligible within the analysis.
Calibration
• Frequency Internal calibration and
accuracy = Daily
• Precision: Once in a month

• 5.1 Operate the instrument as per respective

Standard Operating Procedure.
• 5.2 Switch ‘ON’ the instrument.
• 5.3 Switch ‘ON’ the balance.
• 5.4 The display will blink with 8.8.8.8.8.8.
• After few seconds, the display will show 0.00 g.
• 5.6 If display is not stable, press the TARE key &
wait till the display shows 0.00 g.
• 5.7 Perform internal calibration
Accuracy
• Place 5 gm. on the weighing pan.
• Note the weight.
• Calculate the difference between the
weight in certificate and observed
weight.
• Repeat the above steps using 50gm &
100 gm. weights.
precision
• Place 5 gm. in the weighing pan
• Note the weight
• Repeat the above two steps nine times.
• Record the weight in Anenxure-I .
• Calculate the Standard deviation.
• Calculate the measurement uncertainty
using following equation.
• Measurement uncertainty = 2 X SD /
Actual weight from certificate
Calibration of UV-VIS
spectrophotometer
• Parameters for calibration:
➢Calibration for wavelength accuracy.
➢Calibration for absorbance measurement.
➢Gratings performance or stray light test.
➢Resolution power.
Reference standards for UV VIS
spectrsocpy
 Control of Wavelength
• Weight accurately 1.0 gm of Holmium Oxide and
dissolve it in 1.4 M Perchloric acid solution. Makeup to
25 ml with the same solvent.
• Select the method file of CONTROL OF
WAVELENGTH in the instrument.
• After selecting the file press Reference button for
baseline correction.
• Then fill the Cuvette with 1.4M Perchloric acid and put
in the sample cubicle and press reference to zero.
• After auto zero put the Holmium perchlorate solution
in sample cubicle then press start key.
• Scan it and verify the wavelength using absorption
maxima of Holmium Perchlorate solution.
The permitted tolerance for control of
wavelength calibration is given in
below table.
 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 up to 1000 ml with the same solvent.
Mark the solution as (A). (60ppm)
• Place the solution A in sample cell holder.
• Place blank 0.01M sulfuric acid in reference cell
holder.
• Measure the absorbance values at wavelength
235nm,257,313 and 350nm
•
absorption maxima of Potassium Dichromate
solution at a different wavelength and calculate
the absorbance, tolerance is given in below table.
3.stray light test
• Gratings performance :
• 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 must be greater than 2 at 198nm.
 4. Resolution power:
• Prepare 0.02%v/v solution of Toluene in Hexane UV
• Record spectrum of a 0.02% v/v solution of toluene in
hexane
• Measure the absorbance of above solution at 266 nm
and 269 nm using Hexane UV as blank solution.
• The ratio of the maximum absorbance at about 269
nm to that at the minimum absorbance at about 266
nm should not be less than 1.5 unless
otherwise specified in the monograph.
Frequency :
3 Months
Calibration of Fluorimeter
Frequency 6 months + 3days
• Prepare 1000ppm solution using reference
standard Quinine sulphate.
• dilute it further to prepare 1,2,3,4,5 ppm
solution using 0.1N H2SO4 solution and
measure the intensity using primary wavelength
366nm.
• Plot a graph using conc at X axis and intensity
at Y axis
• Correlation coefficient should NLT 0.99
• When it is performed?
• Most HPLC systems in pharmaceutical
laboratories are calibrated every 6 to 12 months.
➢ Periods longer than 12 months are not
recommended, while periods shorter than 3
months are deemed un-necessary.
• ➢ It also required after annual maintenance (or)
major repairs though only effected modules, and
not the entire system need to be calibrated.
 Calibration of HPLC

Pump Calibration System Detector

calibration of injector precision calibration

Flow rate Injector Injection Detector Wavelength

accuracy linearity volume linearity accuracy
accuracy
• Calibration of Pump Flow Rate Accuracy:
• ➢ fill all solvent reservoirs with HPLC grade water.
• ➢ Set the flow rate to 0.5 ml/min.
• ➢ Wait for about 15min to stabilize the system and
ensure that the pressure is stable.
• ➢ Insert the outlet tubing into a 10 ml volumetric
flask and start the stop watch simultaneously.
• ➢ Stop the stopwatch when the lower meniscus
reaches the 10 ml mark on the flask.
• ➢ 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.
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
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
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%.
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, 100μg/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.
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).
Flame photometer
• Preparation of Standard Solution:
• Weigh accurately 2.542 g of analytical reagent
grade NaCl and transfer into 1 litre volumetric flask.
Add 1.9g of analytical grade KCl and make upto 1000ml
with double distilled water. This stock solution is
successively diluted further to get working standard
solution of 10, 20,40,60,80 and 100ppm. Keep highest
conc 100 ppm in sample holder and adjust value of
emission as 100. then record emission of sodium and
potassium in flame photometer.
• Indian Pharmacopoeia