Calibration of UV-Vis Spectrophotometer Purpose :
Calibration of UV-Vis Spectrophotometer, The purpose of this SOP is to describe the operation, calibration and cleaning of UV-Vis Spectrophotometer (Shimadzu, UV-1800) used in the quality control laboratory of General block at XX Pharmaceuticals Limited.
Scope :
This procedure is applicable for UV-Vis Spectrophotometer (Shimadzu, UV-1800), installed in the quality control laboratory of XX Pharmaceuticals Limited.
Definitions / Abbreviation:
[][]SOP: Standard Operating Procedure.
[][]QC: Quality Control.
[][]UV-Vis: Ultraviolet and Visible.
Responsibilities:
Sr. Executive/Executive, QC
[][]To ensure that this procedure is followed.
[][]To maintain the records properly as per SOP.
Manager, Quality Control
[][]To ensure that this procedure is kept up to date.
[][]To confirm that the SOP is technically sound and reflects the required working practices.
[][]To arrange training on the SOP to all concerned personnel and to ensure implementation of the SOP after training.
[][]Schedule calibration of the instrument at the defined intervals.
Head of Quality Assurance
[][]To ensure the overall implementation of the SOP.
[][]Approval of the SOP
Procedure:
Precaution(s):
[][]Prior to use, user must ensure that equipment is calibrated.
[][]Handle the measuring cell with care.
[][]Do not forget to turn off the instrument after use or if the next reading will be taken after long period of time.
Operation: Calibration of UV-Vis Spectrophotometer
[][]Switch ‘ON’ the Computer, Printer and Spectrophotometer.
[][]Wait until initialization is completed and the initialization result will be displayed in the outlet screen of the machine, finally green signal will appear in the outlet light-bar.
[][]Double click on ‘UV probe’ icon in the computer to run the software.
[][]Press ‘Connect’ icon to establish communications with the machine and machine will be ready for any type of analysis in Photometric/ Spectrum mode.
Measurement in spectrum mode
[][]Click “Spectrum” icon to perform measurement in spectrum mode.
[][]Click “Method” icon to set the desired parameters according to the requirement as follows:
[][]Click ‘Measurement’ icon then set following steps:
[][]Wavelength Range: Start & End- as required.
[][]Scan Speed : Fast/ Medium/ Slow/ Very slow
[][]Sampling Interval : As required
[][]Scan mode: Single/ Auto
[][]Input desired filename for the respective data to preserve in distinguishable data-path.
[][]Click ‘Instrument parameter’ to set the measuring mode as: Absorbance or transmittance.
[][]Finally press “OK” to resolve the setup, click ‘Close’.
[][]Click ‘ Baseline’ icon followed by ‘OK’ for baseline correction.
[][]Rinse both the cells with respective blank solution & wipe properly the outer surface of the cell with tissue paper. Put the cells in cell holder and close the curvature.
[][]Click “Auto zero” to compensate the blank reading.
[][]Remove one cell (front side) and after proper rinsing with the required sample solutions fill the cell with sample solution, wipe properly the outer surface of the cell with tissue paper & Set sample in the cell holder and finally close the curvature.
[][]Click “START”. The photometer status window will show “Slewing….” and it will be followed by the active reading.
[][]After completion of measurement click ‘Point pick’ for determining the absorbance/ transmittance at definite wavelength or Click ‘Peak pick’ for determining the absorbance/ transmittance at a given wavelength range.
[][]Click ‘Save’ to preserve the data and print the data on respective report format.
[][]Wash the measuring cells properly with purified water and preserve in appropriate place.
Measurement in quantitative mode
[][]Click “Photometric” icon to perform measurement in quantitative mode.
[][]Click “Method” icon to set the desired parameters according to the requirement as follows:
[][]Wavelength type: point
[][]Input required wavelength in ‘Wavelength (nm)’, click ‘Add’ then press Next
[][]Input ‘Type’ as ‘Single point’, Formula as ‘Fixed Wavelength’. In ‘WL1’ activate the required wavelength, then input standard concentration in ‘STD Concentration’, click Next>Next>Next.
[][]Finally in ‘Photometric method’ input ‘filename’ for the respective data to preserve in distinguishable data-path. Also input ‘Title’ if any, user name in ‘Analyst’ and fill-up ‘Comments’ if any.
[][]Press “Finish” to resolve the setup, click ‘Close’.
[][]Rinse both the cells with respective blank solution &swipe properly the outer surface of the cell with tissue paper. Put the cells in cell holder and close the curvature.
[][]Click “Autozero” to compensate the blank reading.
[][]Remove one cell (front side) and after proper rinsing with the required standard solutions fill the cell with standard solution, wipe properly the outer surface of the cell with tissue paper & Set standard solution in the cell holder and finally close the curvature.
[][]In the ‘Standard table’ input the standard description in the column for ‘Sample ID’, Click on ‘WL column’ and finally click ‘Read Std’ icon to measure the result.
[][]Remove the standard solution cell (front side) and after proper rinsing with the required sample solutions fill the cell with sample solution, wipe properly the outer surface of the cell with tissue paper & Set sample in the cell holder and finally close the curvature.
[][]In the ‘Sample table’ input the sample description in the column for ‘Sample ID’, Click on ‘WL column’ and finally click ‘Read Unk.’ icon to measure the result.
[][]Click ‘Save’ to preserve the data and print the data on respective report format.
[][]Wash the measuring cells properly with purified water and preserve in appropriate place.
[][]Click ‘Disconnect’ icon to detach the software from the machine.
[][]Exit from the ‘UV probe’ software by closing the window. Put off the power of equipment, printer and computer.
Calibration of UV-Vis Spectrophotometer:
[][]Calibrate the UV-Vis Spectrophotometer at 6 month’s frequency either by following procedure or as per supplier’s protocol:
Wave length Scanning
[][]Set desired start wavelength i.e. higher end of the desired spectrum and desired wavelength expansion. Scan the desired wavelength.
Control of Wavelengths
[][]Verify the wavelength at 241.15 nm, 287.15 nm, 361.5 nm & 536.3 nm using the absorption maximum of Holmium perchlorate solution (4% w/v solution of holmium oxide in a solution of perchloric acid containing 14.1% w/v of HClO4). The permitted tolerance is ± 1 nm for the ultraviolet range and ± 3 nm for the visible range. Record data in calibration information sheet as per Annexure-I .
Control of Absorbance
[][]Check the absorbance using a solution of potassium dichromate at the wavelengths indicated in (Annexure-I) which gives for each wavelength the exact values and the permitted limits of the specific absorbance. The tolerance for the absorbance is ± 0.01.
[][]For the control of absorbance, use solutions of potassium dichromate which has previously been dried to a constant mass at 130° C. For the control of absorbance at 235 nm, 257 nm, 313 nm and 350 nm, dissolve 57.0-63.0 mg of potassium dichromate in 0.005 M Sulphuric acid and dilute to 1000.0 ml with the same acid. For the control of absorbance at 430 nm, dissolve 57.0-63.0 mg of potassium dichromate in 0.005 M sulphuric acid and dilute to 100.0 ml with the same acid.
[][]Measure at a path length of 1 cm, at the wavelength 235 nm, 257 nm, 313 nm, 350 nm and 430 nm against 0.005 M Sulphuric acid as blank. Record data in calibration information sheet as per Annexure-I.
Limit of Stray light
[][]Detect stray light at a given wavelength with suitable solutions: for example the absorbance of a 12 g/l solution of potassium chloride in a 1 cm cell increases steeply between 220 nm and 200 nm and is greater than 2.0 at 198 nm when compared with water as compensation liquid. Record data in calibration information sheet as per Annexure-II.
Resolution (for qualitative analysis)
[][]Prepare a 0.02% v/v solution of toluene in hexane. Use hexane as a blank. The minimum ratio of the absorbance at the maximum at 269 nm to that at the minimum at 266 nm is not less than 1.5 unless otherwise stated in the monograph. Suitable certified reference materials may also be used. Record data in calibration information sheet as per Annexure-II.
Path length measurement of cell
[][]The tolerance on the path length of the cells used is ± 0.005 cm. When filled with the same solvent, the cells intended to contain the solution to be examined and the compensation liquid must have the same transmittance. If this is not the case, an appropriate correction must be applied.
The cells must be cleaned and handled with care.
[][]Carryout the test once before first use of a new cell.
[][]Affix the label “CALIBRATED “on the equipment if all results are satisfactory. If the equipment found out of calibration limit affix “NOT FIT FOR USE” label and call Supplier.
Cleaning procedure: Calibration of UV-Vis Spectrophotometer
[][]After completion of each analysis, clean the UV-Vis spectrophotometer as follows:
[][]Remove cuvette from the sample compartment.
[][]Remove the solution from the cuvette and wash with respective solvent in which solution is prepared.
[][]Rinse with water and then with methanol and wipe with tissue paper.
[][]Air dry and keep it at dedicated place.
[][]When required, clean the outer surface of the instrument with Isopropyl alcohol and dry it with tissue
paper.
Annexure:
Annexure-I: Calibration Information Sheet for UV-Vis Spectrophotometer (Control of Wavelength and Control of Absorbance)
Annexure-II: Calibration Information Sheet for UV-Vis Spectrophotometer (Resolution, Limit of Stray light)
Annexure-III: Operation Logbook for UV-Vis Spectrophotometer