Operation and Calibration of UV Spectrophotometer
This Document describes (Standard Operating procedure) Sop for Operation and Calibration of UV Spectrophotometer
Sop for Operation and Calibration of UV Spectrophotometer .
I. Purpose & Scope:
- The Purpose of this SOP is to lay down the procedure to lay down a procedure for Operation and Calibration of UV Spectrophotometer, Make -Lab India
- This procedure is applicable for Operation, and Calibration of UV Spectrophotometer used in Quality Control Department
II. Responsibilities:
- All Quality Control personnel’s shall be responsible to follow and implement this SOP of UV Spectrophotometer
III : Introduction and Procedural Part for UV Spectrophotometer :
General cleaning for UV Spectrophotometer :
- Clean the Instrument with a clean dry cloth every day. Occasionally wet cloth dipped in dilute soap solution or 70 % IPA may be used.
- A precaution has to be taken to clean the instrument immediately with dry cloth to remove the moisture.
Basic operation of UV Spectrophotometer :
- Switch “ON” the instrument
- Select the display on Desktop UV Win 5 Software V 5.2.0 & Double click on icon
- Enter the User name & Password then click ok.
- Wait for initializing & Auto check.
- UV Win 5 Software screen will be display.
- 4 Ribbon Display shows as
- Kinetics
- Quantitative
- Spectrum
- Photometry
- Right click on Ribbon as per analysis requirements.
- Single right click on Measure ( R) / or F4
- Click on Parameter Setting ( P)
- Select the parameters as per analysis requirements.
- Select the set Parameters & click on ok.
- Fill the both cuvettes with Blank solution & wipe cuvettes with clean tissue paper. Put in selected sample compartments.
- Click on Auto zero.
- Remove cuvettes & rinse with two times with sample solution & fill the Sample solution, Put in a selected sample compartments.
- Wait for 1 min. for stabilization.
- Click on Start, Absorbance will appear on Selected Sheet.
- Save as data file by click on file ( F), Save ( S) / Ctrt + S
- Data file path go to D drive UV Data Year (for year folder name as 2017, 2018 etc) Month (Jan, Feb etc.) then (Create product/material wise folder name for Product Brand name & for material e.g. montelukast BP & save the test.)
- Fill the details Name & B.No. — ( As per file path )
- Save all parameters by click on file ( F)
- Click on save parameters / F12
- Save parameters as Products Name, B.No.
- Print the save file & attached to analytical record.
- Confirmation for Select Cell click on Measure ( R)
- Display appears Accessories ( A) click on required cell No. red colour dot will appear.
- Click on OK.
Calibration of UV Spectrophotometer :
Calibration Frequency:
- Once in three months and after each maintenance job
- Switch ON the instrument and allow it to warm for 10 minutes follow the above operation procedure for each calibration operation.
- Ensure the cleanliness of the instrument before doing calibration.
- Operate the instrument as per the SOP on operation of Spectrophotometer.
Control of wavelength in UV Spectrophotometer :
control of wavelength check shall be done by using Holmium perchlorate solution
Preparation of 1.4 M Perchloric acid :
Accurately transfer 11.5 ml of Perchloric acid (70 %) in to a 100 ml volumetric flask and dilute to volume with water and mix well.
Preparation of Holmium perchlorate solution :
Weigh accurately 0.4 g of Holmium oxide and transfer in to a 10 ml volumetric flask. Dissolve and dilute up to mark with 1.4 M Perchloric acid.
Scan the Holmium perchlorate solution in the wavelength range of 200 nm to 700 nm.
Note: Suitable certified reference materials i.e holmium perchlorate filter (Readymade) may also be used instead of Holmium perchlorate solution.
It exhibits four maxima as following wavelength.
| 241.15 nm ± 1 nm |
| 287.15 nm ± 1 nm |
| 361.50 nm ± 1 nm |
| 536.30 nm ± 3 nm |
Control of absorbance in UV Spectrophotometer :
Absorbance check shall be done by using Potassium Dichromate solution.
Preparation of 0.005 M Sulphuric acid :
Add 0.27 ml of Sulphuric acid to 50 ml of water and dilute to 1000 ml with water
Preparation of Potassium Dichromate solution:
For Ultra Violet Range:
- Weigh accurately about 57 mg to 63 mg of potassium dichromate (previously dried at 130oC to constant mass, and cool to room temperature in a desiccators over silica gel)and transfer in to 1000 ml dried and clean volumetric flask, dissolve and make up to volume with 0.005 M sulphuric acid. (Scan the solution in the range 400 to 200 nm). (For absorption maxima at 235,257,313,350 nm)
For Visible Range:
- Weigh accurately about 57 mg to 63 mg of potassium dichromate (previously dried at 130oC to constant mass, and cool to room temperature in a desiccators over silica gel) and transfer in to a 100 ml dried and clean volumetric flask, dissolve and make up to volume with 0.005 M sulphuric acid. (Scan the solution in the range 450 to 400 nm ) (For absorption maxima at 430 nm)
- Check the absorbance of solution against 0.005 M sulphuric acid as blank at the following wavelengths and calculate the specific absorbance for each wavelength.
Wavelength |
Specific absorbanceA (1%, 1 cm) |
Maximum tolerance limits |
235 |
124.5 |
122.9 to 126.2 |
257 |
144.5 |
142.8 to 146.2 |
313 |
48.6 |
47.0 to 50.3 |
350 |
107.3 |
105.6 to 109.0 |
430 |
15.9 |
15.7 to 16.1 |
- Calculation:
Obtained Absorbance x 1000
Calculation of specific absorbance = —————————————-
(For maxima at 235,257,313,350nm) Weight of K2Cr2O7 in g x 100
Obtained Absorbance x 100
Calculation of specific absorbance = —————————————-
(For maxima at 430 nm) Weight of K2Cr2O7 in g x 100
Limit of stray light in UV Spectrophotometer :
Limit of stray light at given wavelength shall detected by using 12 g/l solution of potassium chloride.
Preparation of 12 g/l solution of Potassium chloride:
- Weigh accurately about 1.2 g of potassium chloride and transfer in to a 100 ml clean volumetric flask, dissolve and dilute to volume with water.
- Scan shall be done in the range of 190 nm to 225 nm.
- Absorbance of the above solution in a 1 cm cell increases steeply between 220 nm & 200 nm.
- The absorbance of solution at about 198 nm should be greater than 2.0, when compared with water as compensation liquid.
Resolution ( For Qualitative Analysis ) in UV Spectrophotometer :
Preparation of 0.02 % v/v solution of Toluene in Hexane :
Accurately transfer 2.0 ml of toluene in to a 100 ml clean volumetric flask and dilute up to mark with hexane and mix well. Further pipette out 1 ml of the solution in to a 100ml clean volumetric flask and dilute up to mark with hexane. Mix well.
- Take the spectrum of the solution in the range of 250 nm to 280 nm.
- The ratio of the absorbance at the maximum at about 269 nm to that the minimum at about 266 nm should be more than 1.5.
Resolution Power (for derivative analysis) in UV Spectrophotometer :
Preparation of 0.02 % v/v solution of Toluene in Methanol :
Pipette out 2.0 ml of toluene in to a 100 ml clean volumetric flask and dilute up to mark with methanol. Further pipette out 1 ml of the solution in to a 100 ml clean volumetric flask and dilute up to mark with methanol.
- Take the spectrum of the solution in the range 260 nm to 270 nm. A small negative extremum located between two large negative extrema should be observed. Maxima at 261 nm and 268 nm should be clearly visible.
- The ratio of absorbance at 261 nm and 268 nm should not be less than 0.2.
Cuvette Qualification for UV Spectrophotometer :
- Measure the transmittance of each cuvette at 200nm, 220nm & 240 nm against air blank. % Transmittance should be more than or equal to 80%.
- The absolute difference of % Transmittance between two cuvette at each wavelength should be within 1.5%.
- Enter the details of calibration of instrument as per annexure-I.
- If the calibration of instrument is ok, then affix the calibration tag. If the calibration of instrument is out of limit, then affix the label “OUT OF CALIBRATION”.
IV: Annexure :
Annexure I : Calibration Record
ANNEXURE-I
Calibration Record
| Name of Instrument : UV Spectrophotometer | Date of Calibration : |
| Make : | Model : |
| Department: Quality Control | Instrument ID No : |
| Next due Date : | |
1) Cleanliness of the Instrument:
Observation: Clean / Not Clean Date:
Corrective action in the case of non-compliance: Sign:
2) Any other maintenance:
Observation: Required / Not required Date:
Corrective action in case of non-compliance: Sign:
3) Control of wavelength in UV Spectrophotometer :
Preparation of Holmium Perchlorate Solution:
| Analytical balance ID No. : | |
| Batch Number of Holmium Oxide used : | |
| Batch Number of Perchloric Acid used : | |
| Weight of paper with Holmium Oxide : | |
| Weight of Paper : | |
| Weight of Holmium Oxide : |
Weight accurately _ _ _ _ _ _ gm (0.4 gm) of Holmium Oxide and transfer in to a _ _ ml (10 ml) volumetric flask. Dissolve and dilute up to mark with 1.4 M Perchloric acid and scan in the wavelength range of 200 nm to 700 nm
Note: Suitable certified reference materials i.e holmium perchlorate filter (Readymade) may also be used instead of Holmium perchlorate solution.
| Observed wavelength for absorption maxima | Acceptance criteria |
| 241.15 nm ± 1 nm | |
| 287.15 nm ± 1 nm | |
| 361.50 nm ± 1 nm | |
| 536.30 nm ± 3 nm |
Remark : Complies / Does not comply
ANALYZED BY : CHECKED BY
DATE : DATE:
4) Control of absorbance in UV Spectrophotometer :
A) For Ultra Violet Range: (For absorption maxima at 235,257,313,350 nm):
| Analytical balance ID No. : | |
| Batch Number of Potassium Dichromate used : | |
| Batch Number of Sulphuric Acid used : | |
| Weight of paper with Potassium Dichromate : | |
| Weight of Paper (Tare wt.) : | |
| Weight of Potassium Dichromate : |
Weigh accurately _ _ _ mg (about 57 mg to 63 mg) of potassium dichromate (previously dried at 130oC to constant mass, and cool to room temperature in a desiccators over silica gel) and transfer in to _ _ _ _ml (1000 ml) dried and clean volumetric flask, dissolve and make up to volume with _ _ _ _ M (0.005 M) sulphuric acid. . (Scan the solution in the range 400 to 200 nm)
B) For Ultra Visible Range: (For absorption maxima at 430 nm):
| Analytical balance ID No. : | |
| Batch Number of Potassium Dichromate used : | |
| Batch Number of Sulphuric Acid used : | |
| Weight of paper with Potassium Dichromate : | |
| Weight of Paper (Tare wt.) : | |
| Weight of Potassium Dichromate : |
Weigh accurately _ _ _ mg (about 57 mg to 63 mg) of potassium dichromate (previously dried at 130oC to constant mass, and cool to room temperature in a desiccators over silica gel) and transfer in to _ _ _ _ml (100 ml) dried and clean
volumetric flask, dissolve and make up to volume with _ _ _ _ M (0.005 M) sulphuric acid. . (Scan the solution in the range 450 to 400 nm )
Observation :
| Sr. No. | Wavelength | Absorbance | Specific Absorbance | Maximum Tolerance |
| 1 | 235 nm | 122.9 to 126.2 | ||
| 2 | 257 nm | 142.8 to 146.2 | ||
| 3 | 313 nm | 47.0 to 50.3 | ||
| 4 | 350 nm | 105.6 to 109.0 | ||
| 5 | 430 nm | 15.7 to 16.1 |
Calculation:
- A) For Ultra Violet Range: (For absorption maxima at 235,257,313,350 nm):
Absorbance of Sample x 1000
Calculation of specific absorbance = —————————————-
(For maxima at 235,257,313,350nm) Weight of K2Cr2O7 in g x 100
x 1000
At 235 nm : = —————————– =
x 100
x 1000
At 257 nm : = —————————– =
x 100
x 1000
At 313 nm : = —————————– =
x 100
x 1000
At 350 nm : = —————————– =
x 100
- B) For Ultra Visible Range: (For absorption maxima at 430 nm):
Absorbance of Sample x 100
Calculation of specific absorbance = —————————————-
(For maxima at 430 nm) Weight of K2Cr2O7 in g x 100
x 100
At 430 nm : = —————————– =
x 100
Remark : Complies / Does not comply
ANALYZED BY : CHECKED BY
DATE : DATE:
5) Limit of stray light in UV Spectrophotometer :
Preparation of 1.2 % w/v solution of Potassium chloride:
| Analytical balance ID No. : |
| Batch Number of Potassium chloride used : |
| Weight of paper with Potassium chloride : |
| Weight of Paper (Tare Wt.) |
| Weight of Potassium chloride : |
Weigh accurately _ _ _ _ _ gm (1.2 g) of potassium chloride and transfer in to a _ _ ml (100 ml) clean volumetric flask, dissolve and dilute to volume with water.
Scan the solution in the range of 190 nm to 225 nm in a 1 cm cell using water as compensation liquid.
Observations :
| Wavelength (nm) | Absorbance | Limit : Greater than 2.0 at 198 nm |
| 198 |
Remark : Complies / Does not comply
ANALYZED BY : CHECKED BY
DATE : DATE:
6) Resolution ( For Qualitative Analysis ) in UV Spectrophotometer :
Preparation of 0.02 % v/v solution of Toluene in Hexane:
| Batch number of Toluene used : |
| Batch Number of Hexane used : |
Pipette out _ _ _ ml (2 ml) toluene and dilute _ _ _ _ ml (100 ml) with hexane. Further dilute _ _ _ ml (1 ml) of the solution to _ _ _ ml (100 ml) with hexane (0.02% v/v). Scan the solution in the range of 260 nm to 270 nm using hexane as blank.
Observations:
| Wavelength (nm) | Observed
Absorbance |
Ratio Of absorbance | Acceptance Criteria |
| 269 | More Than 1.5 | ||
| 266 |
Remark : Complies / does not comply
ANALYZED BY: CHECKED BY
DATE: DATE:
7) Resolution Power (for derivative analysis) in UV Spectrophotometer :
Preparation of 0.02 % v/v solution of Toluene in Methanol:
| Batch number of Toluene used : |
| Batch Number of Methanol used : |
Pipette out _ _ _ ml (2 ml) toluene and dilute _ _ _ _ ml (100 ml) with Methanol. Further dilute _ _ _ ml (1 ml) of the solution to _ _ _ ml (100 ml) with Methanol (0.02% v/v). Scan the solution in the range of 260 nm to 270 nm using hexane as blank.
Observations:
| Wavelength (nm) | Observed
Absorbance |
Ratio Of absorbance | Acceptance Criteria |
| 261 | Not Less Than 0.2 | ||
| 268 |
Remark : Complies / Does not comply
ANALYZED BY : CHECKED BY
DATE : DATE:
8) Cuvette Qualification for UV Spectrophotometer :
Measure the transmittance of each cuvette at 200nm, 220nm & 240 nm against air blank. % Transmittance should be more than or equal to 80%. The absolute difference of % Transmittance between two cuvettes at each wavelength should be within 1.5%.
Observations :
| Wavelength (nm) | Observed
Transmittance |
Acceptance Criteria | |
| Cuvette-1 | Cuvette-2 | ||
| 200 | NLT or Equal to 80% | ||
| 220 | |||
| 240 | |||
Remark : Complies / Does not comply
ANALYZED BY : CHECKED BY
DATE : DATE:
Remark: The instrument i.e. UV Spectrophotometer is calibrated and Complies / does not comply with the acceptance criteria
Calibrated By: Checked By: Head QC
Date : Date : Date :
