- OBJECTIVE:
- To lay down the procedure for Calibration of UV-VIS Spectrophotometer, Make – Shimadzu.
- SCOPE:
This SOP is applicable to the procedure for Calibration of UV-VIS Spectrophotometer at {Company Name} {Location}.
- RESPONSIBILITY:
- Executive/Designee Quality Control is responsible to calibrate the instrument as per SOP.
- Head QC: Ensure the compliance of SOP.
- ACCOUNTABILITY:
QA Head shall be Accountable for implementation of SOP.
- PROCEDURE:
A UV-VIS spectrophotometer plays a crucial role in analyzing and characterizing various samples by measuring their interaction with light in the ultraviolet (UV) and visible (VIS) regions of the electromagnetic spectrum.
- Control of Absorbance:
- Dry a quantity of Potassium dichromate (AR Grade) by heating to constant weight at 130°C.
- Take 100mg dry Potassium dichromate in 100ml vol. flask and allow to dissolve in 0.01N H2SO4 dilute up to the mark with 0.01N H2SO4 solution.
- Take 6ml of resulting solution dilute to 100ml with 0.01N H2SO4.
- Measure the absorbance of the solution at 235nm, 257nm, 313nm & 350nm taking 0.01N H2SO4 solution as blank.
- Control of wavelengths:
- Prepare the solution of holmium per chlorate as follows.
- Dissolve 0.5 gm of holmium oxide in 2.4 ml of perchloric acid AR by warming gently and diluting to 10ml with purified water.
- Check the extinction of the solution, permitted tolerance is 1nm for the range 200 to 400 nm and 3 nm for the range 400 to 600 nm.
- Limit of Stray Light:
- Prepare 1.2% w/v solution of Potassium Chloride.
- Measure the absorbance at 200nm.
- Absorbance should be not less than 2.0.
- Resolution Power:
- Prepare a 0.02% v/v solution of Toluene (HPLC grade) in Hexane (HPLC grade).
- Record the ratio of the absorbance at the maxima at about 269 nm to that at the minima at about 266 nm.
- Ratio should not be not less than 1.5.
- Report the result in the format.
- Frequency: Half early.
- Action: If the calibration is not proper then repeat the procedure and report the results to the department head for an appropriate action.
- REFERENCES:
Not Applicable
- ANNEXURES:
| ANNEXURE NO. | TITLE OF ANNEXURE |
| Annexure-I | Calibration record of UV- spectrophotometer |
ENCLOSURES: SOP Training Record.
- DISTRIBUTION:
- Controlled Copy No. 01 : Head Quality Assurance
- Controlled Copy No. 02 : Head Quality Control
- Master Copy : Quality Assurance Department
- ABBREVIATIONS:
| No. | : | Number |
| UV | : | Ultraviolet |
| KOH | : | Potassium Hydroxide |
| HPLC | : | High Performance Liquid Chromatography |
| SOP | : | Standard Operating Procedure |
| QC | : | Quality Control |
- REVISION HISTORY:
CHANGE HISTORY LOG
| Revision No. | Details of Changes | Reason for Change | Effective Date |
| 00 | New SOP | Not Applicable | To Be Written Manual |
Annexure-I
CALIBRATION RECORD OF UV- SPECTROPHOTOMETER
Frequently Asked Question ?
Q: Why is Potassium Dichromate used for absorbance control?
A: Potassium Dichromate is a certified reference material with well-defined absorption peaks at specific wavelengths. Measuring its absorbance at these wavelengths allows us to verify the accuracy of the spectrophotometer’s absorbance readings.
Q: What does “drying potassium dichromate to constant weight” mean?
A: Heating the potassium dichromate removes any moisture it may have absorbed, ensuring the prepared solution has an accurate concentration. Constant weight implies no further weight change with continued heating.
Q: Why are specific dilutions prepared ?
A: The final solution needs to be within a suitable absorbance range for reliable measurement. Diluting a concentrated solution allows achieving this desired range.
Q: What is the purpose of using Holmium Perchlorate for wavelength control?
A: Holmium Perchlorate has sharp emission lines at known wavelengths. By comparing the measured peak positions with the reference values, we can verify the accuracy of the instrument’s wavelength readings.
Q: What does “limit of stray light” refer to and why is it important?
A: Stray light is unwanted light entering the detector besides the main beam passing through the sample. High stray light can lead to inaccurate absorbance readings. Measuring the absorbance of a highly absorbing solution at a wavelength where it should be opaque (like Potassium Chloride at 200nm) helps assess stray light levels.
Q: What does “resolution power” signify in this context?
A: Resolution power reflects the instrument’s ability to distinguish closely spaced peaks. Measuring the absorbance ratio of Toluene in Hexane at specific wavelengths helps evaluate this capability.
Q: How often should calibration be performed?
A: The paragraph suggests “half early” based on internal policy, possibly meaning half the interval between scheduled calibrations. Regular calibration ensures reliable measurements over time.
Q: What happens if the calibration fails?
A: If the results fall outside specified limits, repeating the procedure is recommended. If issues persist, reporting to the department head is crucial for further investigation and potential instrument repair or adjustments.