Calibration of UV spectrophotometer (UV-VIS Spectrophotometer) for all parameters as per IP, BP and USP pharmacopoeia like, Internal Calibration, Match pairing of UV cell / cuvette, Control of wavelength, control of Absorbance, Limit of Stray light, Resolution and Resolution Power, Linearity Study and 2nd derivatives.
UV Calibration / Calibration of UV Spectrophotometer :
Parameters :
- Calibration shall perform for the following parameters.
UV Spectrophotometer - Internal calibration
- Match pairing of cells (Cuvette qualification)
- Control of wavelength
- Control of absorbance
- Limit of Stray Light
- Resolution Power
- Resolution (2nd Derivatives)
- Linearity study
UV Calibration Frequency : Quarterly (± 7 days)
General Procedure for UV Spectrophotometer:
- All calibration standards used for the calibration should prepared as per template / SOP.
- Only use calibration standard which is procured from authentic source with certification and it should..
- NIST traceable or
- Pharmacopoeial reference standards or
- From instrument manufacturer.
- Alternatively suitable commercial certified filters (NIST traceable) may be used.
- If the commercially available filters being used, Refer the values provided by the manufacturer in the certificate for comparison.
Internal Calibration of UV Spectrophotometer :
- Internal calibration should perform for the parameters mentioned in Attachment for Internal calibration observation sheet.
- Perform the internal Calibration as per manufacturer’s instruction.
- After completion of internal calibration the results obtained shall fill in Attachment -Internal calibration observation sheet, the format may differ base on manufacturer’s internal calibration requirement) for further evaluation and conclusion.
Note : Perform the internal calibration of UV Spectrophotometer, if option available in software.
Match Pairing of Cells (Cuvette Qualification):
- Clean the cell with methanol
- Remove any spot if any.
- Then rinse and fill the cell with distilled water.
- Measure its apparent absorbance against air at 240 nm for quartz cells and 650 nm for glass cells.
- The apparent absorbance should not be greater than 0.093 for 1 cm quartz cells (UV region) and 0.035 for 1 cm glass cells (Visible region).
- After that measure the apparent absorbance after Rotate the cell in its holder (180°) again
- Check the absorbance, Rotating the cells should give the absorbance difference not greater than 0.005 from initial.
- Record the observations and attach the printout of UV graphs with calibration template.
Control of Wavelength:
- Reagent Preparation
- 4M Perchloric Acid : 5.74 ml of Perchloric Acid (AR grade, 11.6M) shall take in a clean and dried 50 ml of volumetric flask and volume shall make up to to 50 ml with distilled water and mix well.
- Preparation of 4% w/v solution of Holmium oxide:
- 0.4 gm of Holmium oxide (HO) (AR grade) shall be taken in a clean and dried 10 ml of Volumetric Flask.
- Then add 8 ml 1.4 M Perchloric acid .
- After that heat and sonicate the Flask till dissolve.
- Then make up volume with 1.4 M Perchloric acid and filter the solution through Whatman no.41 paper or Use certified standard solution of 4.0% w/v Holmium oxide.
-
Calibration Procedure :
- Take the UV spectrum of 4%w/v Holmium oxide in 1.4 M Perchloric acid solution from 200 nm to 600 nm against the 1.4 M Perchloric acid as a blank.
- Wavelength shall be check for the peak detection of Holmium Oxide at 241.15 nm, 287.15 nm, 361.5 nm, 486.0 nm and 536.3 nm.
- The permitted tolerance limit shall be ± 1 nm for the range of 200 nm to 400 nm (UV range) and ± 3 nm for the range of 400 nm to 800 nm.(Visible range)
- Record the observations and attach the printout of UV graphs with calibration report.
Control of Absorbance:
-
Preparation Reagent / Dilution
- 0.005 M Sulfuric acid: 0.54 ml sulfuric acid (AR grade, 18.4 M) shall be taken in a clean and dried 2000 ml volumetric flask containing at least 50 ml of distilled water.
- Make up final volume cautiously to the mark with distilled water and mix well.
- Potassium dichromate solution in 0.005 M Sulfuric acid: Stock solution for 430 nm (600 ppm):
- Use potassium dichromate previously dried to constant mass at 130°C and About 60 mg (57 mg-63.0 mg) of potassium dichromate (AR grade) shall take in 100 ml clean and dried volumetric flask,
- Dissolve and make up the volume with 0.005 M Sulfuric acid and mix well or
- Use certified standard solution of potassium dichromate(600 ppm).
- Final solution (60 ppm):
- 10 ml of above stock solution shall taken in 100 ml clean and dried volumetric flask, make up the volume with 0.005 M Sulfuric acid and mix well or
- Use certified standard solution of potassium dichromate (60 ppm).
- 0.005 M Sulfuric acid: 0.54 ml sulfuric acid (AR grade, 18.4 M) shall be taken in a clean and dried 2000 ml volumetric flask containing at least 50 ml of distilled water.
-
Calibration Procedure:
- Take the spectrum of the Potassium dichromate final solution between 200 nm to 400 nm using 0.005 M Sulfuric acid as a blank.
- Measure the absorbance of peak detection at 350 nm & 257 nm and Valley detection at 313 nm & 235 nm.
- Absorbance of the Potassium dichromate stock solution shall taken at 430 nm using 0.005 M Sulfuric acid as a blank in photo-metric mode. Calculate the specific absorbance (A1 cm1%) & verify the results.
-
Calculation formula :-
Control of absorbance = (Absorbance X 10000 ) / Wt. Taken in mg.
Control of absorbance (for λ 430 nm) = (Absorbance X 1000) / Wt. Taken in mg.
-
- Record the observations and attach the printout of UV graphs with calibration report.
Also visit : HPLC Calibration – A Complete Guide
Limit of Stray Light :
-
Preparation Potassium chloride solution: (12,000 ppm):
- 1.2gm of potassium chloride (AR grade) shall take in 100 mL clean and dried volumetric flask.
- Dissolve and make up the volume with distilled water and mix well or
- Use certified standard solution of Potassium chloride solution (12,000 ppm).
-
Procedure:
- Absorbance of the potassium chloride solution shall be taken against distilled water as a blank between 220 nm and 190 nm in scan mode.
- Check absorbance at 198 nm by keeping cursor.
- Absorbance steeply increases between 220 nm to 200 nm and shall ³ 2.0 at 198 nm.
Linearity Study :
- Preparation of 0.005 M Sulfuric acid:
- 0.54 ml sulfuric acid (AR grade, 18.4 M) shall take in a clean and dried 2000 ml volumetric flask containing at least 50 ml of distilled water.
- Make up final volume cautiously to the mark with distilled water and mix well.
Linearity Solution Preparation in 0.005 M Sulfuric Acid :
- 100 ppm Solution:
- Weigh 100 mg of potassium dichromate (AR grade) (previously dried to constant mass at 130 °C ).
- Transfer in 1000 ml in clean and dried volumetric flask,
- Dissolve and make up the volume with 0.005 M Sulfuric acid and mix well or
- Use certified standard solution of Potassium dichromate (100 ppm).
- 80 ppm Solution:
- Take 20 ml of 100 mg/lit solution in 25 ml clean and dried volumetric flask and dilute up to mark with 0.005 M Sulphuric acid and Mix well or .
- Use certified standard solution of Potassium dichromate (80 ppm).
- 60 ppm Solution :
- Take 15 ml of 100 mg / lit solution in 25 ml clean and dried volumetric flask and dilute up to mark with 0.005 M Sulfuric acid and mix well or
- If 60 ppm (conc.) certified standard solution of Potassium dichromate available, same can be used.
- 40 ppm Solution :
- 20 ml of 100 mg/lit solution shall be taken in 50 ml clean and dried volumetric flask and dilute up to mark with 0.005 M Sulphuric acid and mix well or
- If certified standard solution of Potassium dichromate (40 ppm) is available. It can be used directly.
- 20 ppm Solution :
- 20 ml of 100 mg / lit solution shall be taken in 100 ml clean and dried volumetric flask and dilute up to mark with 0.005 M Sulphuric acid and mix well or
- Use certified standard solution of Potassium dichromate (20 ppm).
- After preparing the Solution. measure the absorbance of the solutions at 257 nm by using 0.005 M sulfuric acid as a blank.
- Graph shall be plotted between absorbance verses concentration.
-
Acceptance Criteria:
- R-square value shall £ 0.999.
Resolution Power:
- Preparation of Toluene solution in Hexane (0.02%v/v) :
- Take 2 ml of Toluene (HPLC grade) in 100 ml clean and dried volumetric flask and diluted up to mark with Hexane (HPLC grade).
- Then take 1 ml of this solution is in 100 ml clean and dried volumetric flask and diluted up to mark with Hexane and mix well or
- Use certified standard solution of Toluene in Hexane (0.02%v/v).
- Take the spectrum of the Toluene solution in the range of 255 nm to 275 nm against Hexane as a blank.
- The ratio of the absorbance at the maximum at about 269 nm to that the minimum at about 266 nm NMT than 1.5.
Resolution (2 nd Derivative test):
- Preparation of Toluene solution in Methanol (0.02%v/v) :
- Take 2 ml of Toluene (HPLC grade) in 100 ml clean and dried volumetric flask and diluted up to mark with Methanol (HPLC grade).
- Then further take 1 ml of this solution in 100 ml clean and dried volumetric flask and
- Dilute up to mark with Methanol and mix well or
- Use certified standard solution of Toluene in Methanol (0.02%v/v).
- Take second derivative spectrum of the resulting solution in the range of 255 nm to 275 nm.
- A small negative extremum (or trough) located between two large negative extrema (or troughs) at about 261 nm and 268 nm, should clearly visible, as shown in below figure.
- The ratio A/B ( pl. see figure) is not less than 0.2.
- Solution for calibration of UV Spectrophotometer shall prepare freshly and shall use within 24 hrs. Record the readings.
- If any part of the instrument is replaced during the maintenance then record the activity in the instrument history card and if required, calibrate the instrument.
- If the readings do not fall within the specified ranges, Contact to service engineer .
Re-Calibration :
- After maintenance (change in critical parts like lamp,filter,mirrors etc.). Re-Calibrate the UV spectrophotometer .
- Upon change in cuvette. Perform Cuvette Qualification..
Out Of Calibration :
- In case failure of UV Calibration in any of the its calibration parameter. Follow SOP on handling out of calibration procedure for laboratory instruments.
Relocation of UV Spectrophotometer :
- If the instrument is shifted for any purpose. Perform the Qualification.
- After following maintenance and for other maintenance. Carryout the UV Calibration ,
- Decide the Calibration requirement case by case basis.
Attachments / Observation Tables for UV Calibration Data Recording
Match pairing of cells (Cuvette qualification)
Control of wavelength
Control of Absorbance
Limit of Stray Light
Resolution Power
Resolution (2nd Derivatives)
- Take the second derivative spectrum of the sample in the range of ____________________ 255 nm-275 nm using _____________ methanol as a __________ blank.
- Calculate the ratio A/B as given in figure below,
A small negative extremum at _________ nm, found between two large negative extrema at________ nm and ___________ nm.
Ratio A/B = ____________ = ___________ (Limit: NLT 0.2)
Linearity study
UV Calibration – Conclusion / Result Table :
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