SOP Title: Operation, Cleaning and Calibration UV-Visible Spectrophotometer

SOP Title: Operation, Cleaning and Calibration UV-Visible Spectrophotometer

1. Objective:

To lay down the procedure for Operation, Cleaning and Calibration of UV-Visible Spectrophotometer.

 

2. Scope:

This SOP is applicable for Operation, Cleaning and Calibration of UV-Visible Spectrophotometer located in Quality Control Laboratory.

 

3. Responsibility:

• Quality Control Department: To prepare and review the SOP. To follow the procedures laid down for Operation, Cleaning and Calibration of UV-Visible Spectrophotometer as per this SOP.
• Engineering Department: To carry out preventive maintenance as per schedule and procedure defined.
• Quality Assurance Department: To review and approve the SOP and Annexures.

 

4. Accountability:

Head Quality Control Department, Head Quality Assurance Department

5. Procedure:

5.1		Safety Precautions/Instructions:
5.1.1		Ensure all the electrical connections are properly earthed. Ensure the power plug of the UV-Visible Spectrophotometer is fixed in the socket properly.
5.1.2		Wear personal protective equipments such as safety glasses, hand gloves, nose mask during operation of instrument.
5.1.3		Ensure that the ON/OFF switch is working properly by switching “ON” the UV-Visible Spectrophotometer.
5.2		Operational  Procedure:
5.2.1		Check the validity of Calibration before starting any operations.
5.2.2		Switch ON the instrument.
5.2.3		The spectrophotometer performs various checks and initialization settings.
5.2.4		The time required for initialisation is around 4-5 minutes.
5.2.5		Wait for the initialization of the instrument.
5.2.6		After normal completion of the initialization procedure the Main Menu will appear on the screen.
		Photometric Absorbance/Transmittance Mode:
5.2.7		To select the Photometric MEASUREMENT, press “1”.
5.2.8		To select Absorbance/Transmittance by Pressing the  key toggles between Absorbance and Transmittance.
5.2.9		Set the wavelength [ l ] key. Enter the wavelength required and press [ENTER] key.
5.2.10		Put the blank solution in both the cuvettes and place them in the cell holder compartment.
5.2.11		Press the key [AUTO ZERO]. Blank will automatically give zero absorbance / Transmittance.
5.2.12		Open the compartment and remove the first cuvette. Put the test solution in first cuvette and place it in the cell holder in the sample compartment.
5.2.13		Read the Absorbance/ Transmittance from the screen. Press (PRINT) to take print of Absorbance/ Transmittance.
5.2.14		Press the [RETURN] key to return to the preceding screen.
5.2.15		Remove both the cuvettes, discard the solution and wash with purified water and then rinse with acetone.
5.2.16		Scanning Range: Set the range of the wavelength scan. Enter the scan starting and ending wavelength in that order. The wavelength input is 190 nm to 1100 nm and the starting wavelength must be the longer wavelength of the two. The minimum scan range is 1 nm (Scan start wavelength) to greater than or equal to 1 nm (Scan end wavelength).
5.2.17		The data sampling interval is determined from the scanning rate (1) and the wavelength range, so that the number of data pieces that are scanned by a single run is 1,000 minimum.
5.2.18		As the data sampling interval becomes larger, the number of data pieces becomes smaller, which means that the practical scanning rate becomes faster.
5.2.19		No. Of scans: Set the number of times a scan will be repeated. If this is set to 2 or More times, the interval setting will be displayed with the scanning repetitions on the same display line. When you press the START/STOP key once, measurement will be repeated only the set number of times.
5.2.20		Display mode: This parameter will toggle between Sequential <-> Overlay each time this item is selected.
5.2.21		Sequential: The screen is renewed for each scan and only the spectrum from that measurement is displayed.
5.2.22		Overlay: The screen is not updated with each scanning result. The spectrum display for each scan is left as it is so that multiple spectra are overlaid in the display.
5.2.23		Gain (E mode only): This will be displayed only in the E (energy) mode. This sets the gain for the photometer amplifier so that measurement can be performed without saturation of the energy curve.  The minimum setting is 1 and the maximum is 4. Sensitivity is increased by approximately 4 times with each level.
5.2.24		Light Source: (E mode only): This will be displayed only in the E (energy) mode. Select a light source for the energy measurement. The selected light source will be used regardless of the parameter setting for the light source.
5.2.25		W lamp: Selects the tungsten iodine lamp (halogen lamp) as the light source.
5.2.26		D2 lamp: Selects the deuterium lamp as the light source.
5.2.27		OFF: Turns OFF both of the W and D2 lamps. The light source mirror turns to the third light source.
5.2.28		Put the blank solution in both the cuvettes and place them in the cell holder in the Compartment.
5.2.29		Select the Meas. Mode, Scanning range, Rec. range, Scan speed etc as per requirement.
5.2.30		Press the ‘F1’ key for [Base Corr.] base line correction.
5.2.31		After competition of base line correction. Open the compartment and remove the first cuvette.
5.2.32		Put the test solution in first cuvette and place it in the cell holder in the sample compartment.
5.2.33		Press the START/ STOP Key. The measurement will be started with the measurement screen displayed.
5.2.34		To stop the measurement, press the STRAT/ STOP Key again. After the measurement has been finished (stopped), the measurement screen will be displayed.
5.2.35		The measurement result can be printed out as a hard copy of the screen on the printer (optional) by pressing the PRINT KEY. Pressing the RETURN key will return to the parameter configuration screen.
5.2.36		After reading, take out both the cuvettes from the sample compartment.
5.2.37		Press the [RETURN] key, to return to the preceding screen and switch OFF the instrument.
5.2.38		Update the “Instrument/Equipment Usage Log Book” as per SOP.
5.3		Cleaning Procedure:
5.3.1		Wash both the cuvettes with distilled water and then dry with methanol. Place them back into the cuvette case.
5.4		Calibration Procedure:
		Frequency:  Quarterly or after carrying out any major maintenance work.
5.4.1		Generate the A. R. No. On the “Instrument/Equipment Calibration Register” as per SOP number and give requisition to QA Department for calibration formats of instruments on the due date of calibration.
5.4.2		For issuance of calibration report refer SOP.
5.4.3		Perform the calibration of instrument as per Annexure and procedure mentioned below.
5.4.4		Calibration of UV-Visible Spectrophotometer is performed for below parameters: 1. Control of Wavelength 2. Control of Absorbance 3. Limit for Stray Light 4. Resolution 5. Derivative Spectrophotometry 6. Cell Check
		1. Control of Wavelength:
5.4.5		Control of Wavelength is done by using Holmium Perchlorate solution
	5.4.6	Preparation of Holmium Perchlorate solution: Dissolve 0.400 gm of Holmium Oxide in 6 ml of 1.4 M Perchloric acid solution and dilute to 10 ml with 1.4M Perchloric acid solution.
	5.4.7	Preparation of 1.4 M Perchloric acid solution: Dilute 5.74 ml of Perchloric acid to 50 ml with purified water.
	5.4.8	Repeat the procedure given for operation under ‘Spectrum Mode’.
	5.4.9	Read the absorbance of solution in spectrum mode from 200 nm to 400 nm and 400 nm to 600 nm, using 1.4M Perchloric acid solution as blank.
	5.4.10	Check the wavelengths at 241.15 nm, 287.15 nm, 361.50 nm, 486.00 nm and 536.30 nm.
	5.4.11	Enter the details in calibration report as per Annexure. Ensure that the results obtained, comply with the acceptance criteria.
		2. Control of Absorbance:
	5.4.12	Control of Absorbance is done by using Potassium Dichromate.
	5.4.13	Preparation of Potassium dichromate solution: For UV range: Dissolve 0.060 gm of Potassium Dichromate previously dried to constant weight at 1300C in 500 ml of 0.005M sulphuric acid and dilute to 1000 ml with 0.005M sulphuric acid. For Visible range: Dissolve 0.060 gm of Potassium Dichromate previously dried to constant weight at 130°C in 50 ml of 0.005M sulphuric acid and dilute to 100 ml with 0.005M sulphuric acid.
	5.4.14	Repeat the procedure given for operation under ‘Spectrum Mode’.
	5.4.15	Take the spectrum scan with Meas. Mode ® absorbance (ABS), Scanning range® 220 nm to 460 nm, Rec. Range ® 0.200 to 0.980 Absorbance and scan speed ® medium or measure the absorbance at specified maximum wavelengths.
	5.4.16	Take out the print and note the Absorbance at 235 nm, 257 nm, 313 nm, 350 nm and 430 nm and calculate A (1%, 1cm) using the formula given below.
	5.4.17	Calculation for UV range: –    Absorbance x 1 x 1000 Weight of  K2Cr2O7 in gm x 100
	5.4.18	Calculation for Visible range: –    Absorbance x 1 x 100 Weight of  K2Cr2O7 in gm x 100
	5.4.19	Enter the details in calibration report as per Annexure No. 01. Ensure that the results obtained, comply with the acceptance criteria.
		3. Limit for Stray Light:
	5.4.20	Limit for stray Light is detected by using 1.2 % w/v solution of Potassium Chloride (KCl) in distilled water with a path – length of 1cm at 200 nm when compared with purified water as reference liquid.
	5.4.21	Limit for stray Light should be more than 2.0.
	5.4.22	Enter the details in calibration report as per Annexure No. 01. Ensure that the results obtained, comply with the acceptance criteria.
		4. Resolution:
	5.4.23	Record the spectrum of a 0.02%v/v solution of Toluene in Hexane.
	5.4.24	Preparation of 0.02%v/v solution of Toluene in Hexane: Pipette out 1.0 ml of Toluene in 50 ml dried volumetric flask and dilute to 50 ml with Hexane and shake well (solution A). Pipette out 1.0 ml of (solution A) in 100 ml dried volumetric flask and dilute to 100 ml with Hexane (Solution B).
	5.4.25	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.
	5.4.26	Enter the details in calibration report as per Annexure No. 01. Ensure that the results obtained, comply with the acceptance criteria.
		5.      Derivative Spectrophotometry:
	5.4.27	Record the spectrum of a 0.02 % v/v solution of Toluene in Methanol in the range 255 nm to 275 nm, using methanol as blank.
	5.4.28	Preparation of 0.02 % v/v solution of Toluene in Hexane: Pipette out 1.0 ml of Toluene in 50 ml dried volumetric flask and dilute to 50 ml with methanol and shake well (solution A). Pipette out 1.0 ml of (solution A) in 100 ml dried volumetric flask and dilute to 100 ml with methanol (Solution B).
	5.4.29	A small negative extrema located between two large negative extrema at about 261 nm and at 268 nm should be clearly visible.
	5.4.30	Enter the details in calibration report as per Annexure No. 01. Ensure that the results obtained, comply with the acceptance criteria.
		6.      Cell check
	5.4.31	First make auto zero using air as a reference.
	5.4.32	Measure the absorbance of each cell one by one by filling with methanol at 240 nm.
	5.4.33	Keep the filled cell in sample compartment by keeping reference cell empty.
	5.4.34	Enter the details in calibration report as per Annexure No. 01. Ensure that the results obtained, comply with the acceptance criteria.
	5.4.35	Update the “Instrument/Equipment Usage Log Book”, “Annual Calibration Schedule” and “Monthly Calibration Schedule” as per SOP.
	5.4.36	Affix the “Calibration Status Label” on the Instrument as per SOP
	5.4.37	If calibration activity is outsourced, the values are to be transcribed in the Annexure and external party certificate to be attached.

 

6. Definitions / Abbreviations:

• Definitions:
• Breakdown: Any activity leading to operation of instrument/equipment other than the set parameters or unusual sound or vibration observed in the instrument/equipment.
• Calibration: Tests performed to ensure that the instrument/equipment is functioning as per set parameters. These tests are performed as per predefined frequency and also after any major repair or replacement of parts.
• Preventive Maintenance: Maintenance activity performed to ensure that the instrument/equipment will function smoothly and to avoid breakdowns. These activities are performed as per predefined frequency.

 

• Abbreviations:

Abbreviation	Expansion
UV	Ultra Violet
ml	millilitre
mg	milligram
%	Percentage
T	Transmittance
l	Wavelength
ABS	Absorbance
D2	Deuterium
g	gram
M	Molar
nm	nanometer
0C	Degree Celsius
K2Cr2O7	Potassium Dichromate
w/v	Weight by volume
v/v	Volume by volume
F	Function
A. R. No.	Analytical Reference Number

 

*Note – Ready to use SOP available in “DOWNLOAD” Section.