operation and calibration of uv-visible spectrophotometer
1.0 Purpose
1.1 To describe a procedure for operation and calibration of UV-Visible Spectrophotometer.
2.0 Scope
2.1 This standard operating procedure is applicable for operation and
calibration of UV Visible Spectrophotometer located at Instrument Room in Quality Control Laboratory at ABC Pvt. Ltd.
3.0 Responsibility
Officer QC/ Executive QC Preparation of SOP and follow the procedure as per SOP
Assistant Manager Reviewing and Training of SOP
Manager QC Implementation of SOP
4.0 Accountability
4.1 Manager Quality Control is accountable for implementation and compliance with SOP.
5.0 Procedure
5.1 Operation
5.1.1 Ensure that the instrument is clean and free from dust.
5.1.2 Open the sample compartment and check that there is no cuvette.
5.1.3 Connect the instrument to main power supply. Switch ON the instrument and
wait for initialization set up, the MODE menu is displayed on the screen.
5.1.4 To select “PHOTOMETRIC” mode press the numeric key “1” and wait for configuration set up.
5.1.5 The display shall show wavelength and absorbance /or transmittance.
5.1.6 To select/ change absorbance or transmittance press F1 toggles key.
5.1.7 To set the wavelength press the key “GO TO WL” and then press the numeric
keys corresponding to the required wavelength (given in the STPs), and then
press “ENTER”. Wait for Configuration setup.
5.1.8 As per the entered wavelength, UV lamp and / or Visible lamp shall automatically
activated by the instrument.
5.1.9 Fill both the cuvettes with blank solution, open and place them into sample compartment
close the compartment door, after few seconds the initial absorbance shall be displayed.
5.1.10 Press the key “AUTOZERO” and wait till 0.0000 is appeared on the screen. Remove
the front cuvette and fill it with the reference (standard) solution, as per the standard testing
procedure. Place it in the compartment and close the chamber. Instrument will display the
absorbance/ transmittance value. Note down the reading. Repeat the same for sample solution.
Press print to print the report, if required.
5.2 Spectrum mode Operation
5.2.1 Press the “RETURN” key followed by “MODE” key. Mode menu is displayed (if Screen is not on mode menu).
5.2.2 Press numeric key “2”, spectrum mode shall be displayed on the screen.
5.2.3 To change measuring mode, press and toggle numeric key “1” to select absorbance (Abs),
Transmittance (%T) or energy (E) measurement. Select Absorbance mode.
5.2.4 Press the numeric key “2” and set the scanning range
(e.g. for scanning range 600-199 nm press 6,0,0 followed by ENTER & 1,9,9 followed by ENTER, respectively.
5.2.5 Press the numeric key “3” and set the absorbance recording range.
Set the recording range 0.00 to 2.00 by pressing numeric keys followed by ENTER similarly as step 5.2.4.
5.2.6 Press the numeric key “4” to select scanning speed. Feed the scanning speed by
pressing key “1” for very fast, “2” for fast, “3” for medium, “4” for slow, “5” for very slow,
followed by ENTER. Normally select “3” for medium scanning speed, unless otherwise specified.
5.2.7 Press the numeric key “5” to select number of scans & press ENTER and select the
required intervals (e.g. for 60 sec, press keys 6,0 and Enter.
5.2.8 Press the numeric key “6” to select Sequential or Overlay mode.
5.2.9 Press F1 key, the instrument shall perform base line correction automatically.
5.2.10 Fill both the cuvettes with Blank solution place in the sample chamber. Press “AUTOZERO” to set zero.
5.2.11 Now remove front cuvette, fill with the sample to be scanned, place in the sample
compartment an close the lid of the chamber. Press “STOP/START” key.
5.2.12 The instrument shall start scanning and scanned spectra between the specified wavelengths
shall be displayed on screen, after completion of scanning it gives the “beep” sound. Move the
cursor continuously by pressing the arrow keys left to right or vice-versa. Check and note the
absorbance at specified wavelength maxima. (i.e. at the cursor point, absorbance shall be displayed on the top of the screen).
5.2.13 Press “PRINT” key to print the spectrum.
5.2.14 To take print without cursor press “PRINT”, the system will ask either Copy or Draw.
For Copy (without cursor) press “F3.
5.2.15 For Draw, press “F4” and select Frame type by pressing “F2” or “F3” or “F4”.
5.2.16 Press Function key F4 to save the data or else press “F2” (Save) then enter file number and
select name by pressing curser key then press “F1” (End).
5.2.17 To cancel the data press “RETURN” key then “MODE” key.
5.2.18 Press Function key F4 to save the data or else press F2 to cancel the data.
5.3 Operation Utilities Mode
5.3.1 Press numeric key “9”. The UTILITIES MODE menu shall be displayed on the screen.
5.3.2 Press the numeric key “F3”. The maintenance menu shall appear on the screen.
5.3.3 To check the performance, press the numeric key “1” and wait and press “ENTER”
The instrument shall check its performance parameters automatically, and checklist shall be displayed on the screen.
5.3.4 Press, “RETURN” key followed by “MODE” key, the mode menu shall be displayed.
5.3.5 As the work is over remove the cuvettes, clean and Switch OFF the instrument.
5.3.6 Transfer the rinsed and used sample carefully into a beaker allocated for waste to avoid the
spillage on the floor and thrown out the waste into washbasin with a stream of water.
5.3.7 Report any discrepancy observed during operation of the instrument to Manager-QC/QA or
his authorized representative, and notify the defect to Engineering Department/ Service Engineer to
rectify the defect. Affix ‘Under Maintenance’ label on the instrument.
5.4 Calibration (Frequency: after 6 Months)
5.4.1 Calibration of UV-VIS Spectrophotometer is done by Indian Pharmacopoeia as four steps
5.4.1.1 Control of Wave length
5.4.1.2 Control of Absorbance
5.4.1.3 Limit of Stray Light
5.4.1.4 Resolution Power
5.4.2 Control of Wave length
5.4.3 Weight accurately 0.400 g of Holmium Oxide and dissolve it in 1.4 M Perchloric acid solution.
Make up to 10 ml with the same solvent.
5.4.4 Then fill the Cuvette with 1.4 M Perchloric acid and put in the sample cuvette and press reference to zero.
5.4.5 After auto zero put the Holmium perchlorate solution in sample cuvette then scan between 600 nm to 200 nm.
5.4.6 Scan it and verify the wavelength using absorption maxima of Holmium Perchlorate solution.
5.4.7 The permitted tolerance is given in below table
| S. No. | Maxima Wave length (nm) | Tolerance (nm) |
| 1. | 241.15 nm | 240.15 nm to 242.15 nm |
| 2. | 287.15 nm | 286.15 nm to 288.15 nm |
| 3. | 361.50 nm | 360.50 nm to 362.50 nm |
| 4. | 536.30 nm | 533.30 nm to 539.30 nm |
4. 536.30 nm 533.30 nm to 539.30 nm
5.4.8 Take the printout and documented the activity in Annexure-I.
5.5 Control of Absorbance
5.5.1 Dry a quantity of the Potassium dichromate by heating to constant weight at 1300C.
5.5.2 Weight accurately about 60 mg (57 mg to 63 mg) of dried potassium dichromate and
dissolve it in 0.005M sulphuric acid solution. Make upto 1000ml with the same solvent. Mark the solution as (A).
5.5.3 Weight accurately about 60 mg (57 mg to 63 mg) of dried potassium dichromate and
dissolve it in 0.005M sulphuric acid solution. Make up to 100 ml with the same solvent. Mark the solution as (B).
5.5.4 Then fill the Cuvette with 0.005 M Sulphuric acid for blank and put in both sample cubicle and press reference to zero.
5.5.5 After auto zero put the Potassium Dichromate solution labeled as solution ‘A’ in sample cubicle then press start key taking absorbance individually for first four wavelength mentioned in ‘Table I’.
5.5.6 Now take absorbance at 430 nm for solution ‘B’.
5.5.7 Note the absorption maxima of Potassium Dichromate solution at different wave length and
calculate the absorbance, tolerance is given in below table.
| S. No. | Wavelength (nm) | Absorbance E (1%1cm) | Maximum tolerance |
| 1. | 235 | 124.5 | 122.9 to 126.2 |
| 2. | 257 | 144.5 | 142.8 to 146.2 |
| 3. | 313 | 48.6 | 47.0 to 50.3 |
| 4. | 350 | 107.3 | 105.6 to 109.0 |
| 5. | 430 | 15.9 | 15.7 to 16.1 |
5.5.8 Limit of Stray light
5.5.8.1 Dry a quantity of the Potassium chloride by heating to constant weight at 1300C.
5.5.8.2 Weight accurately 1.20 g of dried potassium chloride and dissolve it in 50 ml distilled water.
Make up to 100 ml with the same solvent.
5.5.8.3 Then fill the Cuvette with water for blank and put in both sample cubicle and press reference to zero.
5.5.8.4 Check the absorbance of above solution using water as a blank at 200 nm.
5.5.8.5 Absorbance should be greater than 2.0.
5.5.9 Resolution power
5.5.9.1 Prepare 0.02% v/v solution of Toluene in Hexane UV.
5.5.9.2 Then fill the Cuvette with Hexane for blank and put in both sample cubicle and press reference to zero.
5.5.9.3 Measure the absorbance of above solution at 266 nm and 269 nm using Hexane UV as blank solution.
5.5.9.4 The ratio of absorbance maxima at 269 nm to that of 266 nm minima should be more than 1.5.
5.5.9.5 Document the activity in Calibration Record of UV-VIS Spectrophotometer Format Annexure I.
5.5.10 Cuvette Cleaning procedure
5.5.10.1 If any strain found in cuvette.
5.5.10.2 Dip the cuvette in 0.1M Nitric Acid for 30 minutes.
5.5.10.3 Wash the cuvette with purified water.
5.5.10.4 After water washing wash the cuvette with organic solvent (Methanol or Acetone).
5.5.10.5 After dry keep the cuvette in cuvette box.
6.0 Reference
Indian Pharmacopoeia
7.0 Attachments
Annexure I : Calibration Record of UV-Visible Spectrophotometer
Annexure II : Log book of wash water U.V. Spectrophotometer
8.0 Distribution
Distribution of the controlled copy to concerned departments.
Additional copies may be issued for specific purpose.
9.0 Abbreviations
QC : Quality Control
SOP : Standard Operating Procedure
UV : Ultra Violet
g : Gram
% : Percentage
Nm : Nano Meter
mg : Miligram
10.0 Revision History
| Revision No. | Supersedes | Effective Date | Details of Revision |
| 01 | Nil | New SOP |
Annexure I
CALIBRATION RECORD OF UV-VISIBLE SPECTROPHOTOMETER
Instrument ID. No. ………………………. Instrument Make ……………………… Model No. …………………….. Date of calibration:…………………….
Next due date of calibration…………………
(1) CONTROL OF WAVELENGTH Balance No. ………………………
Weight of Holonium oxide ……….g (0.400 g) dissolve in 10 ml of 1.4 M perchloric acid
| S. No. | Maxima at wavelength | Observed Maxima of Holmium oxide |
Tolerance
|
Test Result |
| 200 to 400 nm | ||||
| 01 | 241.15 nm | 241.15 nm ± 1 nm |
Pass/Fail |
|
| 02 | 287.15 nm | 287.15 nm ± 1 nm | ||
| 03 | 361.50 nm | 361.50 nm ± 1 nm | ||
|
01 |
400 to 600 nm | Tolerance |
Pass/Fail |
|
| 536.30 nm | 536.30 nm ± 3 nm | |||
(2) CONTROL OF ABSORBANCE
Method (i) : Solution (A) Balance No. ……………………….
Weight of Potassium Dichromate (Previously dried at 130 C)………mg (60 mg) dissolves in 1000 ml of 0.005 M Sulphuric Acid
| Wave length | Absorbance | E1 % 1 cm | Set E1 % 1 cm | Tolerance | Test Result |
| 235 nm | 124.5 | 122.9 to 126.2 | Pass/Fail | ||
| 257 nm | 144.5 | 142.8 to 146.2 | Pass/Fail | ||
| 313 nm | 48.60 | 47.0 to 50.3 | Pass/Fail | ||
| 350 nm | 107.3 | 105.6 to 109.0 | Pass/Fail |
Method (ii) : Solution (B) Balance No. …………………….
Weight of Potassium Dichromate (Previously dried at 130 C)………mg (60 mg) dissolves in 100 ml of 0.005 M Sulphuric Acid
| Wave length | Absorbance | E1 % 1 cm | Set E1 % 1 cm | Tolerance | Test Result |
| 430 nm | 15.9 | 15.7 –16.1 | Pass/Fail |
- LIMIT OF STATY LIGHT Balance No. ………………………
Weight of Potassium Chloride … … … gm (1.2 gm) dissolves in 100 ml of water.
| Sr. No. | Wavelength in nm | Absorbance observed | Acceptable limit | Test Result |
| 1 | 200 nm | Not less than 2.0 | Pass/Fail |
(4) Resolution power:
0.02% v/v solution (1 ml→100 ml→1 ml→50 ml) of toluene in Hexane UV.
The ratio of the absorbance at the maximum at about 269 nm to that at the minimum at about 266 nm is not less than 1.5.
| Sr. No. | Wavelength in nm | Absorbance observed | Ratio | Acceptable limit | Test Result |
| 1 | Maximum
269 nm |
Not less than 1.5 | Pass/Fail | ||
| 2 | Minimum
266 nm |
Solvent/Reagent Uses
| S. No. | Solvent/Reagent | Lot No | Make |
Conclusion: Instrument meets/does not meets the acceptance criteria and is calibrated/not calibrated for use.
Calibrated by: Checked By:
Date: Date:
