Calibration of Instruments

Calibration of Instruments


At the end of this session, the student will be able to

       Outline the concept of calibration

       Explain the calibration of various analytical instruments



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


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


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


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



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.


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.


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.


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.


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.


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


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.


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.

Post a Comment