1. Calibration of UV-Visible Spectrophotometry

2. When/Where it is Required
Calibration
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

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.
A = e c t

5. Parameters to be calibrated
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 Procedure: Chemicals required: 1.4% Hclo4, Ho2O3
Hclo4 + Ho2O (Ho(ClO4 )3 ) (Holmium perchlorate solution)
Measure the absorbance of both (Ho(ClO4 )3 ) and Hclo4.

7. Acceptance Criteria Deviation results in S.No. Max.Wavelength (nm)
Tolerance (nm) 1.
241.1 2.
287.15 3.
361.5 4.
536.3

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

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

10. Control of Absorbance 57-63mg K2Cr2O7 in 1000ml + 0.005M H2So4
Measure the absorbance at 235nm, 257nm,313nm & 350nm using 0.05M H2So4 as reference.
Formula for calculating % of absorbance

11. Acceptance Criteria S.No. Max.Wavelength (nm) A (1% 1cm)
Tolerance (nm) 1.
235
124.5 2.
257
144.0 3.
313
48.6 4.
350
106.6

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