Calibration of UV-Visible Spectrophotometer

Calibration of UV-Visible Spectrophotometer

 

 

1. Control of Wavelength: 

Preparation of 4% w/v solution of Holmium Perchlorate:

Weighed ____________ gm (0.4) of Holmium Oxide in _______ ml (10) volumetric flask and added about ______ ml (6) of 1.4 M Perchloric acid and made the volume to ________ ml (10) with 1.4 M Perchloric acid.

Read the absorbance of solution in Spectrum mode from 200nm – 400nm and 400nm – 600nm using 1.4 M Perchloric acid as blank  solution for baseline  correction.

Stated Wavelength	Observed Wavelength	Acceptance Criteria
241.15 nm		240.15 – 242.15 nm
287.15 nm		286.15 – 288.15 nm
361.50 nm		360.50 – 362.50 nm
486.00 nm		483.00 – 489.00 nm
536.30 nm		533.30 – 539.30 nm

 

2. Control of Absorbance: 

3. A) For UV Range (235 nm, 257 nm, 313 nm, 350 nm)

  Preparation of Potassium Dichromate Solution:

(Dry a quantity of potassium dichromate powder by heating at 130^oC for 3Hours)

Weighed _____________ mg (between 57.0 mg to 63.0 mg) of previously dried potassium dichromate powder and dissolved in 0.005M Sulphuric acid to produce _________ ml (1000).

 

  Preparation of 0.005M Sulphuric acid:

Added slowly with stirring _______ ml of sulphuric acid to about __________ ml of Water.

 

 Read the absorbance of solution in Spectrum mode in 200 nm – 400 nm using 0.005M Sulphuric acid as

blank solution for baseline correction, as indicated in table below.

 

Abs. of soln. x 1 x 1000

Calculation = ———————————————-  = —————

Wt. in potassium dichromate gm x 100

 

1. B) For Visible range (430 nm)

Weighed ______________ mg (between 57.0mg to   63.0mg) of previously dried potassium dichromate powder and dissolved in 0.005M Sulphuric acid to produce ________ ml (100). Read the absorbance of solution at about 430 nm using 0.005M Sulphuric acid.

Abs. of soln. x 1 x 100

Calculation = ———————————————– = —————–

Wt. in potassium dichromate gm x 100

 

Wavelength	Absorbance	A (1%,1cm)	Acceptance Criteria
235 nm			122.9 – 126.2
257 nm			142.8 – 146.2
313 nm			47.0 – 50.3
350 nm			105.6 – 109.0
430 nm			15.7 – 16.1

 

3. Limit of Stray Light:

Stray light is detected by using a solution of 1.2%w/v potassium chloride at a path-length of 1cm at about 200 nm by using water as blank.

Weighed ______________ (1.2) gm of potassium chloride and dilute to ________ (100) ml with water.

Absorbance at 200 nm =________ (Acceptance Criteria: Not less than 2.0)

 

4. Resolution Power:

   Preparation of 0.02%v/v solution of Toluene in Hexane:

Pipette out _________ ml (1) of Toluene in  dried volumetric flask and diluted to _________ ml (50) with Hexane (solution A) .

Pipette out _________ ml (1) of (solution A) in dried volumetric flask and diluted to ________ ml (100) with Hexane (Solution B) .

Record the spectrum of 0.02% v/v solution of toluene in hexane (Solution B).

1.   a) Absorbance at the maximum at about 269 nm = _________.
2.   b) Absorbance at the minimum at about 266 nm = _________.
3.  c) The ratio of the absorbance at the maximum at about 269 nm to that at the minimum at about 266 nm =_____________.         (Acceptance Criteria: Not Less than 1.5)

 

5. Derivative Spectrophotometry:

    Preparation of 0.02 % v/v solution of Toluene in Methanol:

Pipette out __________ ml (1) of Toluene in dried volumetric flask and diluted to ________ ml (50) with methanol (solution A).

Pipette out _______ ml (1) of (solution A) in dried volumetric flask and diluted to _________ ml (100) with  Methanol (Solution B).

Record the spectrum of 0.02% v/v solution of Toluene in Methanol (Solution B).

     Observation:   _____________________________________________________________________________________________________________

 

Acceptance Criteria: A small negative extrema (or trough) located between two large negative extrema (or troughs) at about 261 nm & 268 nm should be clearly visible.

 

 

6. Cell Check

Absorbance of First Cell (At 240 nm with Methanol): ____________

Absorbance of Second Cell (At 240 nm with Methanol): ___________

(Difference between Absorbance of First and Second cells):

Cell 1 Absorbance	Cell 2 Absorbance	Difference	Acceptance Criteria
			± 0.005

Reference Material Details:

Sr. No.	Name	Batch No.	Valid upto
1.	Holmium Oxide
2.	Potassium Dichromate
3.	Potassium Chloride
4.	Toluene

 

The results Complies / Does not complies as per acceptance criteria.

 

Performed By: ________________ Checked By: ______________

Date: ________________                    Date: ______________

 

“End of Document”