Operation and Calibration of Photofluorometer

  1. Objective

To lay down a procedure for operating and calibration of Photofluorometer. It is used to determine the assay of Vitamin B1 & Vitamin B2.

  • Scope

This SOP is applicable for operation and calibration of Photofluorometer (Make: Systronics, Model 152)in Quality Control.  

  • Responsibility

QC Chemist / Officer : Follow the procedure and maintain record as applicable.   

  • Accountability

Head Quality Control  : System Compliance

  • Procedure:  
    • Operation:
      • Ensure that equipment is clean.
      • Check the calibration due date.
      • Set the sensitivity control in LOW position, STANDARD and BLANK (coarse & fine) controls to minimum i.e. counter clockwise.
      • Insert appropriate primary & secondary filters in their respective slots.
      • Switch ON the instrument. Check the running of cooling fan. This can be noticed by slight noise from the fan and the flow rate of the air from the perforated sheet at the back.
      • Set SENSITIVITY switch control in high position.
      • Do not press the SHUTTER, adjust display to zero by BLANK CONTROLS (coarse & fine).
      • Return SENSITIVITY switch control in low position. Readout should remain zero with in ± 1 digit.
      • Insert test tube of standard. Depress the SHUTTER and adjust STANDARD controls (in conjunction with SENSITIVITY control) till display reads standard reading or 100 counts for full scale. Always adjust 100 full scale at the lowest possible sensitivity. In case 100 full scale is not possible at LOW sensitivity, try at MEDIUM and if still not possible at MEDIUM try at HIGH. Call this reading “S”. Do not disturb BLANK controls.       
      • Replace the standard solution with the blank solution. Depress the SHUTTER and note the display reading “B”.
      • Replace the standard solution with the unknown solution. Depress the SHUTTER and note the display reading “U”.
      • Calculate the concentration of unknown solution using the formula given below:


                Concentration of unknown: ———- x  concentration of standard      


  • After completion of work clean the test tubes & place properly.
    • Switch off the equipment.
    • Enter the details in Instrument log (Annexure I of SOP No. QC-042).
    • Calibration:
      • Operate the equipment.
      • Dissolve 100 mg Quinine Sulphate in 0.1 N Sulphuric Acid and dilute to 1000.0 ml with same solvent (Stock Solution).
      • Further dilute to make the dilution 1, 2, 3, 4, & 5 ppm in 0.1 N Sulphuric Acid and measure the intensity using primary wavelength 366nm.
      • Plot a graph by taking concentration at Y axis & intensity at X axis.
      • Acceptance Criteria:  The correlation coefficient should be NLT 0.99.
      • Enter the details.
      • Prepare the calibration tag and affix it on the equipment.
      • Enclose the old calibration tag with the record and don’t throw it.
      • If the variation is not within the specified tolerance limits:
        • Label the equipment with a tag, “OUT OF CALIBRATION”.
        • Inform the Department Head.
        • Do not use equipment until all errors are rectified. After rectification, again calibrate the equipment & record the observation.
      • Frequency: 6 Months ± 3 days.
      • Important:
        • On the equipment before 30 minutes prior to use.
        • Both arrows (one on OPTICS block & other on the filter holder) should be matched, when inserting filters in their respective slots.
        • For more details refer instrument manual.
        • In case of any deviation inform to service engineer.
  • Annexure(s)


  •  Reference(s)

Manufacturer manual

Chapter  No. 2.4.5,   (IP-2015).

  • Glossary           

SOP                :           Standard Operating Procedure

QA                   :           Quality Assurance

QC                  :           Quality Control

nm                  :           Nenometer

NLT                 :           Not less than

ppm                :           Part per million

  • Revision History
Rev. No. Details of changes Reason for change

Bhanu Pratap Singh


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