# Calibration of Instruments

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
dummy cuvette from sample holder & close the lid.

Ø  By
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.

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

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.