Calibration of UV-Spectrophotometer (2017)

PROCEDURE :
PRECAUTIONS: Place the instrument as far as possible away from any strong magnetic or electric field or any electrical apparatus generating a high frequency. Avoid placing it where severe vibrations exist or when the sun beats. Place the instrument free from dust and corrosive gases. To extend the life of the source lamp, have them powered only when required. Cells should be cleaned and handled with great care. Wipe the cell properly with tissue or dry soft cloth. Never use alkali, abrasive / etching material or hot concentrated acids.
OPERATIONS: Switch on main switch. When the instrument power is turned ON, UV-Spectrophotometer the starts executing various checks and initializations.
CALIBRATION: CONTROL OF WAVELENGTHS: Preparation Of 1.4 M Perchloric Acid Solution: Take 11.5 ml of Perchloric acid (about 60%w/w strength) into a clean   100 ml volumetric flask. Dilute to the volume with distilled water(Note :Dilute 8.5 ml to 100 ml with  distilled water in case of 70% Perchloric acid) Preparation Of Holmium Perchlorate (With Holmium Oxide): Weigh accurately about 400 mg Holmium oxide. Transfer into a 10 ml volumetric flask containing 5 ml of 1.4 M Perchloric acid solution. Shake the solution well to dissolve Holmium oxide and make up the  volume to the mark with 1.4 M Perchloric acid solution.                                                                                           Procedure For Control Of Wavelengths: Take a pair of cuvette having a path length of 1 cm.  Perform the base line correction with 1.4 M Perchloric acid solution from 200 to 600 nm. Remove the cell from sample compartment. Rinse the sample cell with Holmium perchlorate solution, fill the cell with the same, clean the smooth surfaces of the cell with tissue paper and place it in the sample compartment. Wavelength Maximum Tolerance 241.15 nm 240.15 to 242 .15 nm 287.15 nm 286.15 to 288.15 nm 361.50 nm 360.50 to 362 .50 nm 536. 30 nm 533.30 to 539.30 nm
CONTROL OF ABSORBANCE:  Preparation Of 0.005 M Sulphuric Acid: a)     Take a clean 1000 volumetric flask and fill to the mark with distilled water by adding 0.27 ml Conc. Sulphuric acid to the above flask. b)     b)  Take a clean 100 volumetric flask and fill to the mark with distilled water by   adding 0.027 ml Conc. Sulphuric acid to the above flask. Preparation Of K2Cr2O7 Solution: Take Analytical Reagent grade K2Cr2O7 (potassium dichromate) in to a    weighing bottle and heat at130oC in a laboratory oven for 2 hours  and  Cool it in a desiccator.  Solution A:  weigh accurately  quantity not less than 57.0 mg  and not more than 63.0 mg and dissolve in 1000 ml volumetric flask containing 300 ml of 0.005 M Sulphuric acid.  Shake the flask well to dissolve Potassium dichromate completely. Make up the volume with 0.005 M Sulphuric acid. Solution B : weigh accurately  quantity not less than 57.0 mg and not more than 63.0 mg and dissolve in 100 ml volumetric flask  containing 50 ml of 0.005 M Sulphuric acid. Shake the flask well to dissolve Potassium dichromate completely. Make up the volume with 0.005 M Sulphuric acid. Procedure For Control Of Absorbance:  Take a pair of  cuvette having a path length of 1 cm. Perform base line correction with 0.005 M Sulphuric acid from 200 to 600 nm. Remove the cell from sample compartment. Rinse the sample cell with solution A, fill the cell with the same, clean the smooth surfaces with tissue paper. Place the cell in sample compartment, scan from 200 to 600 nm and measure the   absorbances at 235 nm, 257 nm, 313 nm and 350 nm. Rinse the sample cell with solution B, fill the cell with the same, clean the smooth surfaces with tissue paper. Place the cell in sample compartment, measure the   absorbances at  430 nm. Acceptance Criteria: Calculate the specific absorbance using the following equation.         Specific absorbance at x nm    =  Absorbance at x nm X 1000 X 10                                                                             weight of sample(mg)  The specific absorbances against each wavelength should be as follows : Wavelength (nm) Specific absorbance (1%, 1 cm) Maximum tolerance 235 124.5 122.9 to 126.2 257 144.5 142.8 to 146.2 313 48.6 47.0 to 50.3 350 107.3 105.6 to 109.0 430 15.9 15.7  to 16.1
LIMIT OF STRAY LIGHT: Preparation Of 1.2% W/V Potassium Chloride Solution Take about 3 gm of Potassium chloride dry at 250oC for about 1 hours. Weigh accurately about 1.2 g of Potassium chloride. Transfer into a 100 ml volumetric flask containing 75 ml distilled water. Shake the flask well to dissolve completely and make up to the volume with water. Procedure for limit of stray light Take a pair of cuvette having a path length of 1 cm. perform the auto zero with distilled water.                Remove the cell from sample compartment. Rinse the cell in the potassium chloride solution, fill the cell with the same, clean the smooth surfaces with tissue paper and place it in  sample compartment. Measure the absorbance at 200 nm. Acceptance Criteria The absorbance should be more than 2.
RESOLUTION POWER: Preparation of 0.020 % v/v of toluene in Hexane solution:   Dilute 2 ml of Toluene to 200 ml with Hexane. Further dilute 2 ml of above solution to 100 ml with Hexane. Record the spectrum in the range 260 to 275 nm of 0.020 % v/v toluene in Hexane using Hexane in the reference cell. Calculate the ratio of the absorbance at the maximum at about 269 nm to that at the minimum at about 266 nm Acceptance Criteria : Ratio is not less than 1.5.
SPECIFICATION OF CELLS: In `Photometry mode ’ Select transmittance mode. Set the wavelength at 200 nm. Then press `auto zero’ to get 100 % transmittance with air blank in both the cell holders. Then place the cell in the sample compartment filled with distilled water using air blank and read the transmittance.  Similarly repeat the operation for 220 nm and 240 nm. Repeat the test  for second cell. Acceptance Criteria: The transmittance values should meet the requirement as given in the following table. Wavelength % Transmittance 200  nm 73 to 77 % 220  nm                                83 to 87 % 240  nm                                85 to 89  %
LINE FLATNESS:    With air blank in the sample and reference compartment, perform the test.      Scan in the wavelength range 220 to 700 nm.  Obtain the spectrum in the % transmittance mode. Acceptance Criteria: The line obtained should not deviate from a horizontal line at any point by more than 98.0% - 102.0%  transmittance.
SOLVENTS: Take the absorbance of following solvents at about 254 nm with reference to distilled water.          a)   Ethanol (95%)          b)   Ethanol          c)   Methanol          d)   Cyclohexane Acceptance Criteria: Absorbance should not exceed 0.10