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To calibrate absorbance scale and detect stray light in

an UV/Vis spectrophotometer.

Theory:Calibration is the process of constructing a mathematical model to relate the response from an analytical instrume

to the properties of the samples .Hence, the validity of the number generated by a spectrophotometer shou

occasionally be verified by the measurements of materials with known spectral properties.

A spectrophotometer is capable of both transmitting and receiving light. The instrument is used to analy

samples of test material by passing light through the sample and reading the intensity of the wavelengths. Differe

samples impact the light in different ways allowing a researcher or technician to learn more about the materials the test sample by seeing how the light behaves as it passes through the sample. Hence, Spectrophotometr

calibration is necessary to confirm that the results are accurate.

According to Beer-Lambert law:

A = log10 1/T = log10 I0/I = a1%

1cm * b * c.

For a qualitative/quantitative method based on UV/Vis spectrophotometry, it is required to select the mo

appropriate sample and instrumental conditions for the test with regard to the nature of the sample being analys

and the purpose of the test. So that, accuracy and precision of spectrophotometric measurement depends on t

choice of sample conditions(solvent, concentration and pathlength) , instrumental parameters and

performance(Absorbance scale, wavelength, stray light, resolution power, spectral slitwidth etc.)

Instrumental parameters and its performance should be assessed and controlled regularly with the standa

procedure or according to the manufacturers protocol.

Chemicals required:1)Potassium dichromate(A.R), 2)Potassium chloride(A.R).

Apparatus required:1)UV/Vis Spectrophotometer, 2)Volumtric flasks(1L,100ml).

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Procedure:1.Absorbance scale:

Dry potassium dichromate(A.R.) at 130C to constant weight. Accurately weigh 0.057-0.063 g of potassiu

dichromate and transfer into a 1litre volumetric flask. Shake to dissolve in 0.005M sulphuric acid. Dilute to th

mark with the same solvent. Measure the absorbance of the resulting solution a 235nm, 257nm, 313nm, 350nm

For the absorbance at 450nm, dissolve 0.057-0.063g of potassium dichromate in 0.005M sulphuric acid and dilu

to 100ml in the same solvent. Calculate specific absorbance(A1%

,1cm) for each wavelength.

2.Detection of stray light:

Dry potassium chloride(A.R.) at 105C to constant weight. Weight accurately 1.2g and transfer into a 100ml V.

,dissolve and dilute to the mark with water. Measure the absorbance at 200nm which should be greater than 2

Any absorbance less than 2 indicates the presence of stray light.

Observation:1.Absorbance scale.

Absorbance at 235nm= , 257nm= ,313nm= ,350nm= ,450nm=

Calculation:

Relation:

A1%

,1cm at 235nm: ,at 257nm: ,at 313nm: ,at 350nm: ,at 450nm:

2.Detection of stray light:

Stray light at 200nm =

Results:1.Absorbance scale:

S.No. Wavelength(nm) Specific absorbance(A %,1cm)

required

Specific absorbance(A %,1cm)

calculated

1 235 124.5 (122.9 to 126.2)

2 257 144.0 (142.8 to 145.7)

3 313 48.6 (47.0 to 50.3)

4 350 106.6 ( l04.9 to 108)

5 450 15.9 (15.7 to 16)

2.Stray light at 200nm( requirement 2.0) =