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 %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 λ486.1 + 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 & λsetting from 655 to 657nm.

Ø If the maximum %T is obtained at a λ656.2 + 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 λ485nm 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 K₂Cr₂O₇as 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 λmax 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.