Calibration of UV Spectrophotometer

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 Calibration
    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).
  • 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)

UV Calibration - Control of Wavelength

Control of wavelength

UV Calibration - Control of Wavelength

Control of Absorbance

UV Calibration - Control of Absorbance

Limit of Stray Light

UV Calibration - Limit of Stray Light

Resolution Power

UV Calibration - 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

Linearity study

UV Calibration – Conclusion / Result Table :Conclusion - Result

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