Calibration of UV-VIS Spectrophotometer (PERKIN ELEMER LAMBDA 25)

This document details the procedure for the Operation and Calibration of the UV-VIS Spectrophotometer (PERKIN ELEMER LAMBDA 25)

It is the policy of pharmaceuticals that the written procedure shall be followed for the operation and calibration of UV-VIS Spectrophotometer and its use monitored to ensure that the instrument gives consistently accurate and reproducible results in compliance with regulatory requirements.

This procedure is to be applied at the time of the Operation and Calibration of the UV-VIS spectrophotometer.


Persons along with their responsibilities & account-abilities are given below:

S. No     Designation        Responsibility

01 Executive -        Corporate Quality Assurance            To prepare SOPS

02 Trainee Analyst/ Jr. Analyst/ Analyst/ Sr. Analyst         To follow the SOP accordingly

03 Manager – Quality Assurance               To ensure implementation of SOP.

04 Manager      -          Quality

Control to ensure implementation of SOP.

05 Manager -           Technical-      To ensure implementation of SOP.

06 VP          -           Technical-          To ensure implementation of SOP.

07  Manager- Corporate Quality Assurance-         To ensure implementation of SOP.



5.1.1 Check and ensure that the instrument is clean and suitable for starting the operation.  If it is not, clean and ensures suitability. 

5.1.2 Instrument should be located at the ambient temperature.   

5.1.3 Ensure instrument and printer are connected to an uninterrupted power supply.

5.1.4 Check calibration status of the instrument before use.

5.1.5 Report any discrepancy observed during calibration of instrument to Section Incharge or his representative for corrective and preventive action.

5.1.6 Section In-charge or his representative will report the same to the Manager – Quality Assurance and notify the defect to service engineer to rectify the defect. 

5.1.7 Affix ‘Under Maintenance’ label on the instrument.


5.2.1 Never use a brush to clean the inside of the cuvette.

5.2.2 Clean the Cuvette using Purified water or any organic solvent (e.g. Acetone or Methanol) & dry the cuvette

5.2.3 Add about 1ml of the solution to be measured. Tilt and turn the cuvette so that the solution has contact with all the surfaces. Discard the solution and repeat this rinse once more.

5.2.4 Fill the cuvette about ¾ full of the solution you wish to test.

5.2.5 Wipe the outside of the cuvette with a lint-free, soft tissue to remove any moisture or fingerprints from the outside surface. 


5.3.1 Do not stare into the path of ultraviolet rays path.

5.3.2 Do not touch the fine surfaces, always use the rough surface of the cells to hold.

5.3.3 Ensure cleanliness of the cells before and after use. 

5.3.4 Use Purified water to rinse the cells.

5.3.5 Do not use directly Purified water to rinse cells after use of solutions prepared with immiscible solvents, use ethanol or methanol first and then water.

5.3.6 Use a lint-free cloth duster for wiping the cells.

5.3.7 Use keeping cells in one direction in the cell holder throughout the test to minimize the error in the blank and sample solutions.

5.3.8 Do not shake the solutions of samples immediately before measurement.

5.3.9 Ensure the lid of the UV compartment is closed properly.

5.3.10 The temperature of all solutions/reagents used in the test should not differ by more than 0.5 ºC.

5.3.11 The solvent in the reference cell should be of the same batch as that used to prepare the solutions.


5.4.1 Before starting check power supply is on.

5.4.2 Switch on Instrument by using On/Off present on the rear side of UV-VIS Spectrophotometer.

5.4.3 Allow the UV lamp to glow for mins.

5.4.4 Start Perkin Elmer UV by double-clicking the Icon Perkin Elmer UV Win Lab software.

5.4.5 Insert User name and Password. 

5.4.6 Click Ok

5.4.7 Each time you start the instrument application (by double-clicking the instrument icon from the main window) an Instrument option will start.

5.4.8 Click on the Instrument option it will display Lambda 25 folder

5.4.9 Click on Lambda 25 folder it will display Manual Control

5.4.10 Click Manual Control it will display Settings

5.4.11 Click Settings and set Wavelength as per requirement.

5.4.12 Click Optional Mode and set Absorbance or Transmittance as per requirement.

5.4.13 Enter Wavelength at which you want to perform the Auto zero and then Click Ok. the Instrument goes to the selected wavelength.

5.4.14 From Actions selects Auto zero   

5.4.15 Fill the cuvettes with blank / Solvent solution, wipe with lint-free cloth duster and place it in both the cell holders & press Auto Zero the current wavelength will automatically be set to `0’ Abs. (100% T)

5.4.16 then removes the front side cuvette, discards the blank solution. Rinse the cuvette with the sample solution. Then fill the cuvette with sample solution & replace it in the cell holder. The monitor will display the Absorbance of the sample solution at the wavelength displayed.


5.5.1 The Data collection page allows you to define settings such as scan range, ordinate mode and slit width 

5.5.2 for scan first click icon Methods (by clicking the software)

5.5.3 Click New, open the file

5.5.4 Select New Method

5.5.5 Enter the name of the method and required text in the description field.

5.5.6 Click Scan press key next again next then click save & finish press OK. 

5.5.7 Click Data collection

5.5.8 Click Scan setting

5.5.9 Following Scan settings are available on the Data collection page Start End Ordinate Mode Slit Width Scan speed   Data Interval   Number of cycles   Cycle as fast as possible   Cycle time UV Lamp on UV Lamp off Lamp change at (nm)

5.5.10 Enter sample information such as sample id, description as per requirement by clicking  sample information in the data collection file

5.5.11 Click Auto zero by placing blank in the cell holder

5.5.12 Press Start a plot of wavelength Vs Absorbance will be displayed

5.5.13 Save the scan method by pressing save the result.

5.5.14 Print this as per your requirement

5.5.15 before Exit press files this will display option Exit. Press exit 

5.6 Maintain Logbook of UV-VIS Spectrophotometer on APL/CQA/SOP-042/FR-01 Logbook  for UV-VIS Spectrophotometer (Appendix I)

Calibration of UV-VIS Spectrophotometer




6.2.1 Dilute 11.5 ml of Perchloric acid (70 % w/w) to 100 ml with Purified water (1.4 M HClO4)

6.2.2 Dissolve 0.4 g of Holmium oxide in 5 ml of 1.4 M Perchloric acid with the aid of heating on a water bath, cool, and dilute to 10 ml with the same solvent. (4% w/v Holmium Perchlorate solution).

6.2.3 Alternatively, a ready-made solution can also be used.

6.2.4 Scan the solution in the range of 200 to 700 nm using 1.4 M Perchloric acid as a blank. Note down the maxima observed in the Calibration Data Sheet.  Check the maxima observed against the acceptance criteria.

6.2.5 Acceptance criteria:

Wavelength       Maximum Tolerance 

241.15 nm           (240.15 to 242 .15 nm)

287.15 nm           (286.15 to 288.15 nm)

361.50 nm           (360.50 to 362 .50 nm)

536. 30 nm          (535.30 to 537.30 nm)

6.2.6 Record the calibration data on APL/CQA/SOP-042/FR-02 Calibration datasheet.  (Appendix II)


6.3.1 Control of absorbance using potassium dichromate solution :

6.3.2 Procedure: Add 5.4 ml of sulphuric acid in 500 ml Purified water and dilute to 1000 ml with Purified water (Solution A).

6.3.3 Further dilute 100 ml to 2000 ml with Purified water to give a 0.005 M sulphuric acid solution. (Blank)  [Alternatively, a ready-made solution, can also be used.]

6.3.4 Dry Potassium dichromate (A.R. grade) at 130°C to constant weight and weigh a quantity accurately between 57.0 mg and 63.0 mg into a 1000.0 ml volumetric flask. Add 0.005 M Sulphuric acid to dissolve and dilute to the mark. (Solution B)

6.3.5 Also weigh 57.0-63.0 mg of Potassium dichromate  UV into 100.0ml volumetric flask. Add 0.005M sulphuric acid to dissolve and dilute to the mark (Solution C)

6.3.6 Check the absorbance of solution B at the following wavelengths (nm): 235, 257, 313, 350.

6.3.7 Check the absorbance of solution C at wavelength 430nm.

6.3.8 Record the absorbance at each wavelength on APL/CQA/SOP-042/FR-02 Calibration Data sheet (Appendix II).

6.3.9 Calculate the value A  (1%, 1 cm) as per the following formula :

 A (1 %, 1 cm)     =          Absorbance  x   100   x  100/Weight of K2Cr2O7 in mg

6.3.10 Check the value of A at each wavelength against the acceptance criteria given below :

6.3.11 Acceptance criteria:


 (nm)     Specific absorbance A 1 %

1 cm       Maximum tolerance for absorbance

235         124.5     122.9   to   126.2     EP & IP

257         144.0     142.4   to   145.7      I P

                144.5     142.8   to   146.2     EP

313           48.6      47.0   to     50.3     IP & EP

350         106.6     104.9   to   108.2     IP

                107.3     105.6    to   109.0     EP

430         15.9        15.7 to 16.1   IP &EP


6.4.1 Note the absorbance of a 1.2 % w/v solution of Potassium chloride, at a path length of 1 cm, the absorbance increases steeply between 220 nm and 200 nm and is greater than 2 at a wavelength between 198 nm and 202 nm when compared with Purified water as reference liquid. 

6.4.2  Acceptance criteria:  Absorbance should be greater than 2 and transmittance should be less than 1.0% T.

6.4.3 Record the calibration data on the SOP/FR-02 Calibration datasheet.(Appendix II)

6.5 RESOLUTION: (for qualitative analysis)

6.5.1  0.020 % v/v solution of toluene in Hexane:  

 Dilute 2ml of Toluene to 200ml with Hexane. Further, dilute 2 ml of the above solution to 100 ml with Hexane.  

(Note: Use only spectroscopy grade Toluene and Hexane)

6.5.2. Record the spectrum in the range 260 to 275 nm of 0.020 % v/v toluene in Hexane using Hexane in the reference cell.

6.5.3  Calculate the ratio of the absorbance at the maximum at about 269 nm to that at the minimum at about 266 nm.

6.5.4 Acceptance criteria: Ratio of max/min should not be less than 1.5.

6.5.5 Record the calibration data on the SOP/FR-02 Calibration datasheet. (Appendix II).


6.6.1 0.020 % v/v solution of toluene in methanol: Dilute 2 ml of Toluene to 200 ml with Methanol Further dilute 2 ml of the above solution to 100 ml with Methanol.  

 (Note: Use only spectroscopy grade Toluene and methanol)

6.6.2  Record the second derivative spectrum in the range 255 to 275 nm of 0.020 % v/v   toluene in methanol using methanol in the reference

6.6.3 Acceptance criteria: 

Small negative extrema located between two large negative extrema at about 261 and 268 nm should be clearly visible. The ratio of max/min should not be less than 0.2

6.6.4 Record the calibration data on SOP/FR-02 Calibration datasheet. (Appendix II)


6.7.1 In Photometry mode select transmittance mode. Set the wavelength at 200 nm. Then press auto-zero to get 100 % transmittance with air blank in both the cell holders. Then place the cell in the sample compartment filled with purified water using air blank and read the transmittance. Similarly, repeat the operation for 220 nm and 240 nm. Repeat the test for the second cell. The transmittance values should meet the requirement as given in the following table.

6.7.2      Acceptance criteria:

                Wavelength                                       % Transmittance

                200 nm                                                 Not Less Than 73%

                220 nm                                                 Not Less Than 85%

                240 nm                                              Not Less Than 87%

                365 nm                                                 Not Less Than 90%

                Note: Transmittance of one cell is within ± 1.5% of the other cell.

6.7.3 Record the calibration data on APL/CQA/SOP-042/FR-02 Calibration datasheet. (Appendix II)


6.8.1 With an air blank in the sample and reference compartment, perform the test.            

6.8.2   Scan in the wavelength range 220 to 700 nm.

6.8.3 Obtain the spectrum in the % transmittance mode.

6.8.4 Acceptance criteria: The Io line obtained should not deviate from a horizontal line at any point by more than 99.54 - 100.46 % transmittance.               

6.8.5 Record the Calibration Data on SOP/FR-02 Calibration Data Sheet (Appendix II)

6.8.6 Summarize the calibration data on SOP/FR-03 UV-VIS Summary sheet (Appendix III) 


Generate Instrument Calibration number on the calibration datasheet as INSCALXXYYZZZ Where, INS denotes Instrument, CAL denotes Calibration XX denotes year; YY denotes Month ZZZ denotes sequence number.


INS CAL: Instrument Calibration  

IP & EP   :  Indian Pharmacopoeia & European Pharmacopoeia 

>SOP For Operation and Calibration of the UV light Cabinet.


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