for absorbance accuracy, wavelength accuracy, stray light, and baseline flatness.

2. Scope

This SOP applies to all UV-Visible spectrophotometers used for analytical method development, validation, and routine quality control within the organization.

3. Responsibilities

• Analytical Chemist: Performs calibration and records data in the calibration logbook.
• QA Representative: Reviews and verifies the calibration data for compliance.
• Instrument Supervisor: Ensures timely calibration, troubleshooting, and maintenance.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring the calibration of all UV-Visible spectrophotometers is performed as per this SOP and that the records are maintained appropriately.

5. Procedure

5.1 Calibration Frequency

1. Perform calibration quarterly or after any major maintenance.
2. Calibration should also be performed if abnormal results or instrument errors are observed.

5.2 Equipment and Materials Required

• UV-Visible Spectrophotometer
• Certified Holmium Oxide Filter or Solution
• 0.005 M Potassium Dichromate Solution
• Distilled Water (Blank)
• Quartz Cuvettes
• Validated calibration logbook (Annexure-1)

5.3 Calibration Parameters and Acceptance Criteria

1. Wavelength Accuracy: Use holmium oxide filter/solution. Acceptable variation: ±1 nm from certified value.
2. Absorbance Accuracy: Prepare 0.005 M potassium dichromate and measure at 235 nm, 257 nm, 313 nm, and 350 nm. Compare with acceptance limits as per pharmacopeial standards.
3. Stray Light: Use potassium chloride solution. Absorbance at 200 nm should be ≥2.0.
4. Baseline Flatness: Scan 200–400 nm with distilled water. Fluctuation in absorbance should not exceed ±0.01 AU.

5.4 Calibration Steps

1. Switch ON the UV-Visible spectrophotometer and allow warm-up for 30 minutes.
2. Clean cuvettes and ensure they are free from scratches and fingerprints.
3. Zero the instrument with blank (distilled water).
4. Carry out wavelength calibration using the certified holmium oxide standard.
5. Record the peak maxima and compare with certified values.
6. Prepare and measure absorbance of potassium dichromate solution at specified wavelengths.
7. Record stray light absorbance using 1% potassium chloride at 200 nm.
8. Run a full scan for baseline flatness from 200 to 400 nm with blank cuvette.
9. Record all results in the instrument calibration logbook.

5.5 Documentation

1. Fill Annexure-1 with all calibration readings and analyst signature.
2. Submit for QA verification and sign-off.
3. Paste one copy in the instrument area and another in the QA documentation room.

5.6 Out of Calibration (OOC) Handling

1. If any parameter fails, immediately label instrument as “OUT OF ORDER”.
2. Initiate deviation form and perform root cause analysis.
3. Perform recalibration after corrective action and document accordingly.

6. Abbreviations

• QA: Quality Assurance
• OOC: Out of Calibration
• AU: Absorbance Unit
• nm: Nanometer

7. Documents

1. Annexure-1: UV-Visible Spectrophotometer Calibration Log
2. Annexure-2: Deviation Form Template

8. References

• USP General Chapter <857> – Ultraviolet-Visible Spectroscopy
• ICH Q2(R1) – Validation of Analytical Procedures
• Instrument Operation Manual

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Sanjana Iyer	Rahul Deshmukh	Dr. Smita Narula
Designation	Analyst	QA Reviewer	Head – AMD
Department	Analytical Method Development	Quality Assurance	Analytical Method Development

11. Annexures

Annexure-1: UV-Visible Spectrophotometer Calibration Log

Date	Wavelength Accuracy	Absorbance Accuracy	Stray Light	Baseline Flatness	Remarks
01/06/2025	Within ±1 nm	Pass	2.2 AU	±0.008 AU	All parameters within limits

Annexure-2: Deviation Form Template

This form captures date, deviation ID, description, probable cause, corrective action, and QA comments.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
01/06/2025	2.0	Stray light and baseline flatness sections revised	Annual review and regulatory update	Dr. Smita Narula
15/07/2022	1.0	Initial Release	New SOP	QA Head