Calibration of uv vissible spectroscopy

Calibration, Calibration parameters of UV

1. Calibration of UV-Visible Spectrophotometry

2. Calibration 2 Definition Calibration is the act of ensuring that a method or instrument used in measurement will produce accurate results. Calibration is performed using primary reference standard When/Where it is Required specific time period is elapsed . specific operating hours has elapsed. a new instrument. observations appear questionable.

3. Regulation to be followed ISO 9000 GLP GMP 3

4. UV-Visible Spectrophotometry When a beam of light falls on a solution or homogenous media, a portion of light is reflected, from the surface of the media, a portion is absorbed within the medium. 4 A = e c t

5. Parameters to be calibrated 5 Official Parameters (IP) Wavelength Stray Light Resolution Control of Absorbance Spectral Split width Solvents Non official (Standard books) Noise Stability Baseline Flatness Photometric Accuracy Linearity

6. Wavelength Chemicals required: 1.4% Hclo 4 , Ho 2 O 3 Procedure: Hclo 4 + Ho 2 O 3 (Ho(ClO 4  ) 3  ) (Holmium perchlorate solution) Measure the absorbance of both (Ho(ClO 4  ) 3  ) and Hclo 4. 6

7. A cceptance Criteria Deviation results in 7 S.No . Max.Wavelength (nm) Tolerance (nm) 1. 241.1 240.15-242.15 2. 287.15 286.15-288.15 3. 361.5 360.5-362.5 4. 536.3 533.3-539.3

8. Stray light 1.2 % Kcl solution Determine the absorbance using path length of 1cm at 200nm against purified water as blank. 8 Acceptance Criteria Absorbance > 2

9. Resolution 0.02% v/v solution of toulene in hexane Measure the absorbance at 269nm & 266nm (Hexane as reference) Calculate the ratio 9 Acceptance Criteria Absorbance ratio ≤ 1.5

10. Control of Absorbance 57-63mg K 2 Cr 2 O 7 in 1000ml + 0.005M H 2 So 4 Measure the absorbance at 235nm, 257nm,313nm & 350nm using 0.05M H 2 So 4 as reference. Formula for calculating % of absorbance 10

11. Acceptance Criteria 11 S.No . Max.Wavelength (nm) A (1% 1cm) Tolerance (nm) 1. 235 124.5 122.9-126.2 2. 257 144.0 142.4-145.7 3. 313 48.6 47-50.3 4. 350 106.6 104.9-108.2

12. Solvents 95% Ethanol, ethanol, methanol & Cyclohexane 12 Acceptance Criteria Absorbance at 240nm ≤ 0.1