Calibration of UV-Visible Spectrophotometer
-
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 |
-
Control of Absorbance:
- 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 130oC 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
- 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 |
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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)
-
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).
- a) Absorbance at the maximum at about 269 nm = _________.
- b) Absorbance at the minimum at about 266 nm = _________.
- 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)
-
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.
-
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”