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Calibration of instrumental

methods

Presented by
Dr. A. Suneetha
Dept. of Pharm. Analysis
Hindu College of Pharmacy
Calibration
A set of operations, performed in accordance with a definite
procedure, that compares the measurements performed by an
instrument to those made by a more accurate instrument or a
standard for the purpose of detecting co-relate and reporting,
by adjustment errors met in the instrument tested.

Purpose of Calibration
To ensure readings from an instrument are consistent with other
instruments and to determine the accuracy of the instrument i.e.
that it can be trusted for its observed/displayed measured value.
CALIBRATION OF INSTRUMENTS:
Measurement of Accuracy
 Establishment the relation of an instruments accuracy to the
international standards.

Value of calibration in industry:


 To demonstrate that the industry operates a quality system and
technically competent and are able to generate technically valid results.
 To increase quality & value of product.
 The calibrated measuring instruments (working standards) have the
assurance of an international measuring standards.
Calibration process
The purpose of calibration is to ensure that the measuring
accuracy is known over the whole measurement range under
specified environmental conditions for calibration.

Instrument to be
Instrument Output
Input (whole calibrated
measuring range)

Instrument of The input value


Higher Standard with known
accuracy

Ensure the
Environmental calibration is done
Conditions Standard
Instrument under the specified
(Modifying Inputs) environmental
conditions

Fig.1 Calibration of Instrument


Maintenance and calibration of instruments and apparatus:
 It is primarily responsibility of analyst who operates the
instrument to ensure that the instrument is of appropriate design
adequate capacity to perform intended function consistently and
precisely.
 Ultimate responsibility for accuracy and precision of an
instrument and data it generates is with the analyst using the
equipment.
 It is therefore essential that instruments be inspected cleaned
and adequately maintained.
 Need for calibration and standardization of instruments is well
recognized among scientific community for obtaining analytical
data to be subjected to validation process.
NOTE : No instrument will be used until and unless calibrated or
checked for its performance while under taking the development
of new method.
INSTRUMENT INTERVAL (MONTHS)
UV –visible spectrophotometer 6
Infrared spectrophotometer 4
NMR spectrometer 6
Thermo gravimetric analyzer 6
Polari meter 6
Fluorimeter 4
PH meter 6
Dissolution test apparatus 3
Disintegration test apparatus 6
Ultrasonic water-bath 6
Heating baths 4
Analytical balance 3
HPLC 6
CALIBRATION OF ANALYTICAL BALANCE

Calibrate with internal 20g weight for analytical balance and with
100mg as microbalance.
Analytical balance : Use 20gm weight there should be no variation
in the first four places to the right of decimal .
Micro balance : use 100 mg weight there should be no variation in the
first three places to the right of decimal.

Note : Accuracy of weight(20 gm or 100 mg) is not important aim of


above check is to find out if any drift has been there . Weighing of
fixed weights will determine whether the knife edge is defective or
not if there is no drift there will be no variation in observed weight in
the first four or three places as the case may be.
 Record the weight after the reading is stable for at least few seconds do
not allow the sample to remain on the balance for longer periods as
there can be change in weight due to humidity.

ACCURACY AND PRECISION FOR HIGHER RANGE(110/210gm)


A .Low load test
1.Set the balance for appropriate range 110 or 210 gm
2.Place 10gm weight in middle of pan tare the balance to read 0
3.Add 1gm weight to this pan and record the readings
4.Remove 1gm weight the balance which had earlier been tarred with
respect to 10gm weight should show 0 reading
5.Repeat the step 3 and 4 to get five values
 Calculate mean and standard deviation of five values apply
correction if 1gm weight used differs from the actual standard
weight the balance complies with specifications if the corrected
mean value is 1±0.00021gm its standard deviation of 1.4*10-4 gm.
B. High load test:
1. Place 105 g (100+5) weights on the pan and tare the balance to read
zero. Carry out the steps 2 to 5 as above.
2. The balance complies with specification if the corrected mean value
is 1±0.00033gm with standard deviation of 2.2*10-4 g.

ACCURACY AND PRECISION FOR LOWER RANGE(30/60gm)


Carry out above steps using initially 5g(low load test) or 25(5+20)
(high –load test).
corrected mean values are 1±0.000042 gm for low-load and
1±0.000054g for high load test.
NOTE : Usually the manufacturers provide details about accuracy
and precision of balance which is guiding factor for calibration of
balance.
In general the performance of balance can be checked by using fixed
weight procedure .
CALIBRATION OF UV
SPECTROSCOPY
CALIBRATION OF U.V :
Control of wavelength:
 Verify wavelength scale using absorption maxima of holmium per
chlorate solution the line of hydrogen or deuterium discharge lamp
or lines of mercury vapor arc.
 The permitted tolerance is +/-1 nm for the range 200 to 400nm and
+ /-3 nm for the range 400 to 600 nm.
241.15 nm (Ho) 404.66 nm (Hg)
253.70 nm (Hg) 435.83 run (Hg)
287.15 nm (Ho) 486.00 nm (Dp)
302.25 nm (Hg) 486.10 nm (HP)
313.16 nm (Hg) 536.30 nm (Ho)
334.15 nm (Hg) 546.07 nm (Hg)
361.50 nm (Ho) 576.96 nm (Hg)
365.48 nm (Hg) 579.07 nm (Hg)
Control of absorbance:
Check the absorbance using potassium dichromate solution UV
at the wavelengths indicated in table , which gives for each wave
length the exact value of A( 1%, 1 cm) and the permitted limits.
Wavelength A(%, 1 cm) Maximum
(nm) Tolerance
235 124.5 122.9 to 126.2
257 114.0 142.4 to 145.7
313 48.62 47.0 to 50.3
350 106.6 104.9 to 108.2

Limit of stray light:


Stray light may be detected at a given wavelength with suitable
filters or solutions; for example, the absorbance of a 1.2% w/v
solution of potassium chloride at a path-length of 1 cm should
be greater than 2.0 at about 200 nm when compared with water
as reference liquid.
Resolution power:
When prescribed in a monograph, record the spectrum of a 0.02% v/v
solution of toluene in hexane UV. The ratio of the absorbance at the
maximum at about 269 nm to that at the minimum at about 266 nm is
NLT 1.5 unless otherwise specified in the monograph.

Spectral slit width:


When measuring the absorbance at an absorption maximum the
spectral slit width must be small compared with the half-width of
the absorption band otherwise erroneously low absorbance will be
measured. Particular care is needed for certain substances and the
instrumental slit width used should always be such that further
reduction does not result in an increased absorbance reading.
CALIBRATION OF INFRA-RED
SPECTROPHOTOMETRY
CALIBRATION OF INFRA-RED SPECTROPHOTOMETRY
Infra-red region consists of an optical system capable of providing the
monochromatic light in the region of 4000 to 670 cm-1 (about 2.5 to
15mm) .

Resolution performance of the apparatus:


Record the spectrum of a polystyrene film 0.05 mm in thickness.
The depth of the trough from the maximum at about 2851 cm- 1
(3.51µ m) to the minimum at about 2870 cm-1(3.48µ m) should
be greater than 6% transmittance for prism instruments and 18%
transmittance for grating instruments, and that from the maximum
at about 1583 cm1(6.32 µ m) to the minimum at about 1589cm-1
(6.29µm) should be greater than 6% transmittance for prism
instruments and 12% transmittance for grating instruments.
Verification of the wave number scale
The wave number scale may be verified using a polystyrene film
which has maxima at the wave numbers (in cm-1) shown
3027.1 (± 0.3)* 1583.1 (±0.3)
2924 (±2) 1181.4 (±0.3)
2850.7 (± 0.3) 1154.3 (±0.3)
1944 (± 1) 1069.1 (± 0.3)
1871.0 (± 0.3) 1028.0 (± 0.3)
1801.6 (±0.3) 906.7 (± 0.3)
1601.4 (±0.3) 698.9 (±0.5)
*The numbers in parentheses indicate the accuracy with which these
values have been established.
FTIR Calibration

Calibration:
 Calibration must be performed using a certified polystyrene
film.

 Place the polystyrene film (0.04mm) and scan it between


4000cm-1
to 650 -1cm.

 Control of wave number.

 Check the absorption maxima (transmission minima) at the


following wave numbers in cm-1.
Wave No’s. Tolerance
3060.0 ± 1.5
2849.5 ± 1.5
1942.9 ± 1.5
1601.2 ± 1.0
1583.0 ± 1.0
1154.5 ± 1.0
1028.3 ± 1.0
Resolution:
 Check the resolution performance of the instrument in the
following way.
 Measure difference between percentage transmittance at
transmission maximum of 2870cm-1 and percentage
transmittance at transmission minimum of 2849.5cm-1.
Acceptance criteria: The difference between the
percentage transmittance at transmission maximum
of 2870cm-1 and percentage transmittance at
transmission minimum of 2849cm-1 must be greater
than 18 transmittance.
 Measure difference between percentage
transmittance at transmission maximum of 1589cm-1
and percentage transmittance at transmission
minimum of 1583cm-1.
Acceptance criteria: The difference between
percentage transmittance at transmission maximum
of 1589cm-1 and percentage transmittance at
transmission minimum of 1583cm-1 must be greater
than 12 transmittance.
INTERVAL: Monthly the instrument is calibrated.
CALIBRATION OF HPLC
 The following calibration parameters shall be checked to verify
the performance of the individual components of the HPLC.
The respective instrument should be identified by ‘under
calibration’ label before starting the calibration.

Component Name of the test to be performed


Pump 1. Flow accuracy
2. Gradient accuracy
Injector 1. Injector precision
2. Injector linearity
Detector 1. Detector response linearity
2. Wavelength accuracy
3. Drift and noise
Column oven 1. Temperature accuracy.
Flow rate : 2.0 mL/min
Pump : Gradient
Wavelength : 254nm
Runtime : 30 minutes
Inject : 10µl of water.

COLUMN OVEN TEMPERATURE CHECK


a. Set the column oven temperature to 25, 30, 35, 40, 45°C.
b. Allow the system to attain set temperature.
c. Keep one calibrated working thermometer in column oven and allow
it to attain thermal equilibrium.
d. Ensure that mercury bulb of the thermometer is not touching to any
portion of the compartment.
e. Read the temperature of the thermometer immediately after taking
out.
f. Acceptable range for column oven is ± 2°C of the set temperature.
Large peak response shall be 258nm ± 2nm.
Mobile phase used should be filtered and degassed HPLC grade
menthol as mobile phase.

AUTO SAMPLER INJECTION LINEARITY


a. Freshly prepare the Benzophenone solution 0.10 mg/ml in methanol
and inject 5μl, 10μl, 15μl, 20μl and 25μl in duplicate.
b. From the data plot a graph of injection volume versus area response,
calculate the Correlation co-efficient

Acceptance Criteria: Correlation co-efficient should be not less than


0.99
PROCEDURE FOR INJECTION PRECISION:
Weigh accurately 50mg of drug and dissolve in 50mL of water
(stock).
Dilute 2 mL of the above solution to 100 mL with water (0.02
mg/mL).
Prepare degassed mixture of water and methanol (Ratio 70:30) as a
mobile phase.

Set the following chromatographic conditions


Column : Symmetry C18, 4.6 mm x 75 mm, 3.5 m.
Flow Rate : 1.0mL/min
Column Temperature : 35°C
Wavelength : 273nm
Run Time : 5min
Stabilize HPLC for 30 min.
Inject the solution of 0.02 mg/mL concentration
(injection volume:10µl) 6 replicate injections.
CALIBRATION OF DETECTOR
To a clean, dry 100ml volumetric flask, add 0.1ml of Benzene and
0.1ml of Toluene. Mix by adding mobile phase and make up to
volume. Inject equal quantities three times and record the ratio of the
peak area.
Chromatographic conditions
Detector : 254nm
Flow Rate : 1.0 ml/minute
Mobile Phase (Methanol : Water )80 : 20
Absorbance Scale : 0.02
Attenuation :5
Injection Volume : 20micro liter
Column : C-18 (Shim-pak)
Procedure for wavelength accuracy
 Inject the concentration of 0.02mg/mL (injection volume:
10µl) solution each time with the following parameters by
varying the wavelength from 201nm to 209 nm, 241nm to
249nm and 269nm to 277nm with 1 nm frequency. In case
of PDA, keep the wavelength range from 190nm to 370nm
and extract the spectrum at above wavelength.
 Record accuracy first max, accuracy second max and
accuracy min.
 Acceptance criteria: The accuracy first max should show
205±2nm, accuracy second max should show 273±2nm,
and accuracy min should show 245±2nm.
Procedure for drift and noise
 Keep degassed methanol/water (HPLC grade) 50:50 in channel A
and purge the channel.
 Connect union between pump outlet to detector inlet.
 Stabilize the system for at least 30min with 1.0mL/min for system
cleanup.

The detector should be switched ON at least 2 hours before the


start of testing. Insufficient warm-up may result in drift in excess of
the actual value for the detector.
 Wavelength: 250nm, Flow: 1.0 mL/min, Run time: 70 min.
 Take chromatogram by selecting any 10 minutes range for
‘Noise’ calculation, record maximum absorbance and minimum
absorbance for every minute in the selected range of 10 minutes.
 Take chromatogram, for ‘DRIFT’ calculation, record absorbance
at about 30 minutes and at about 60 minutes.
Calculate the ‘DRIFT/ NOISE’ .
Acceptance criteria:
 Drift  5 x 10-3 AU/Hour
 Noise  1 x 10-4 AU

Note: If the calibration is performed continuously, no further


stabilization is required for injector linearity, detector response
linearity and wavelength accuracy.
Table 1. A typical format for instrument record

Manufacturer Serial
Type of Instrument: Instrument Model:
Number:
Company Serial Company’s Part
Date Introduced:
Number: Number:
Measurement Limits: Location: Calibration frequency:
Person Responsible for
Name: Signature:
Calibration
Instructions for use:
Calibration Record
Calibration Date Calibration Results Calibrated by
REFERANCE:
• Indian Pharmacopoeia, Published by the Controller
of publication,Delhi,Vol 2 – 1996.

• P.D Sethi ,Quantitative Analysis Of drugs in


Pharmaceutical Formulation, pg no.70-75.

• WWW.WIKIPEDIA
THANK Q