Calibration of Instruments
Objective
At the end
of this session, the student will be able to
• Outline the concept of calibration
• Explain the calibration of various
analytical instruments
Validation
Definition:
Ø Validation
is an analytical procedure that deals with the collection of documented
evidence which is established by the laboratory studies
Ø It
contains performance characteristics of the instrument that meet the
requirements for the intended analytical applications
Protocol
Ø It
is a written plan which states how validation will be conducted
Ø This
protocol identifies the
• Equipment
process parameters
• Product
characteristics
• Sampling
data to be collected
• No.
of validation runs and acceptable test
results
Qualification
Ø Qualification
is a part of validation. It is action of proving and documenting the equipment
which are properly installed. Qualification is of 3 types
v Installation qualification
v Performance qualification
v Operation qualification
Installation Qualification
•
It checks the installation site /environment
•
Confirms the specifications of the analytical
instruments.
•
Verifies the conditions of installed equipment.
Operation qualification
•
It includes all procedures and documentation of
instrument
•
When all the procedures are executed & then
it verifies that the system operates to
satisfy the intended purpose
Performance qualification
•
It represents final part of qualification
•
The objective is to ensure that the instrument
is performing within specified limits
Calibration
Ø It
is set of operations which is established under specified conditions
Ø It
is necessary to ensure the accuracy of the data produced during the
process
CALIBRATION OF UV SPECTROPHOTOMETER
SPECTRAL CALIBRATION (visible region)
Ø Ensure
the socket of the power cord is inserted properly. Then switch on the
instrument & allow to stand for 15min to warm up.
Ø Keep
the dummy cuvette in position of sample holder. Set the wavelength to 485nm
& press %T button.
Ø Press
%T in appropriate direction to adjust 0.00 reading on read out. Now remove
dummy cuvette from sample holder & close the lid.
Ø By
adjusting the coarse & fine control set a reading of around 80.0 on read
out. Now set the value of wavelengths in increments of 0.1nm up to 487nm and
read the value of % T at each increment.
Ø Draw
a curve %T Vs λ. If the peak
value of %T is occuring at λ
486.1 + 0.5 nm, the spectral calibration of the instrument in the
visible spectral region is proper.
Ø This
can be confirmed by repeating the above steps with a maximum value of %T of
around 30.0 on the read out & λ
setting from 655 to 657nm.
Ø If
the maximum %T is obtained at a λ
656.2 + 0.5 nm then the spectral calibration of the instrument in the
visible region is confirmed to be proper.
SPECTRAL CALIBRATION (UV region)
Ø Keep
blank filled cuvette & sample (benzene vapour) filled cuvette & set the
λ to 253nm & press the
absorbance button.
Ø Adjust
blank to 0.000 on the read out using coarse and fine adjustment.
Ø Now
place sample into the optical path, value of absorbance of sample at the λ set appears on the read out.
Ø Again
set the values of wavelengths in increments of 0.1nm up to 255nm.Measure the
absorbance at each increment. If the maximum absorbance is observed at 253.9 +
0.5nm the spectral calibration of the instrument in UV region is confirmed to
be proper.
PHOTOMETRIC CALIBRATION
Visible region:
Ø Place
dummy cuvette in sample holder & set %T to “zero”. Now remove cuvette by
using coarse and fine control set & press absorbance button.
Ø If
the maximum absorbance obtained at λ
485nm is 0.398 + 0.002 then the photometric calibration of the
instrument is confirmed to be proper.
Ø To
confirm, repeat above steps, and set 10.0 on read out & again press
absorbance button.
Ø If
the λ at 485nm is 1.000 +
0.002 then it is confirmed the photometric performance in the visible region is
proper.
UV region:
Ø Place
0.1N H2SO4 as blank in cuvette & 60ppm as sample (K2Cr2O7).
Ø Set
exactly to 257nm, if the value of absorbance of sample is 0.864 + 0.005,
the instrument is measuring absorbance properly.
TRANSMITTANCE
Ø As
the value of %T is delivered from absorbance itself, if the instrument is
measuring absorbance properly it is deemed that it measures %T properly.
CONCENTRATION
Ø Place
0.1NH2SO4 as blank & 60ppm K2Cr2O7
as standard & 20ppm 0.1N H2SO4 as sample in cuvette.
Ø Then
press ‘Concentration’ push button and adjust concentration control to 600 for
standard on read out.
Ø Now
place sample holder into optical path, if the value of concentration appearing
on the read out for sample is 200+5, the instrument is measuring
concentration properly.
WAVELENGTH ACCURACY
HOLMIUM FILTER – Record the absorption spectrum from
500 -230nm using slowest scan speed and narrowest slit setting.
•
The λ
max at 453.2, 418.4, 360.9, 287.5, 279.4, 241.5nm.
HOLMIUM PERCHLORATE SOLUTION – Prepare a solution of
Holmium oxide in perchloric acid by warming gently & diluting to 10ml with
water.
•
Record the absorbance from 500 – 230nm.
•
The
wavelength of principal bands should be 485.8, 450.8, 416.3, 361.5, 287.1,
278.7, 241.1nm.
DISCHARGE LAMPS – Place the mercury lamp near the
entrance to the monochromator using minimum slit setting and slowest scan
speed.
•
Record the transmission spectrum from 600
-240nm.
•
The principal emission of mercury are at579.0,
576.9, 546.1, 435.8, 404.5, 364.9 & 253.7nm
LIMIT OF STRAY LIGHT
Ø Weigh
accurately 1.2g of dried potassium chloride in 100ml volumetric flask and make
up to the mark with distilled water.
Ø Measure
the absorbance at 200nm.
RESOLUTION
Ø Prepare
0.02%v/v solution of toluene and make up with hexane. Scan the wavelength from
250 -280 nm.
Ø Maximum
absorbance is 269nm & minimum absorbance is 266nm.