SOP for Operation of Shimadzu UV–1800 UV Spectrophotometer

OBJECTIVE

To lay down a procedure for the operation of a UV Spectrophotometer using UV Probe software. 

SCOPE

This is applicable for the operation of a UV Spectrophotometer using UV Probe software for Shimadzu UV 1800. 

RESPONSIBILITY

It is the responsibility of QC Executives to follow the procedure without deviation. 

ACCOUNTABILITY

Assistant Manager – Quality Control 

Manager – Quality Assurance

PROCEDURE

Basic Operation

• Before starting the analysis, Check and ensure that the instrument is clean & calibrated & is placed on a level stable strong surface. 
• Ensure that the instrument has been connected to the power supply & all communication cords between the units are connected properly. 
• Turn `ON’ the power switch located on the left-hand side of the UV Spectrophotometer. 
• After the instrument initialization has been completed, the mode menu screen will display on the UV Spectrophotometer. 
• Login to the UV Spectrophotometer by pressing enter on the keypad. There is no password. Press “F4” to allow for PC control.

Start up the UV Probe Software

• Double-click the UV Probe icon on the desktop to launch the UV Probe software. 
• The basic window will display. From the toolbar select the required measurements i.e. Photometric, Spectrum, Kinetics, etc. 

Establishing Communication with UV Spectrophotometer

• Click “Connect” on the instrument bar to connect the UV–1800. 
• When communication with the UV-1800 is established the connect button changes to “Disconnect” and the instrument status bar starts indicating the current wavelength & photometric value. To disconnect the communication, click the Disconnect button. 

Basic Mode Menu 

• Photometric: Used to measure the ABSORBANCE or TRANSMITTANCE at the desired wavelength. Photometric mode is for quantitative analysis, measuring absorbance at single wavelengths. 
• Spectrum: Used to scan the wavelength range to measure the absorbance or transmittance of the range selected. Spectrum mode is for wavelength scanning over a continuous wide range. 
• Kinetics: Used to measure the activity from the time-dependent changes in absorbance & % transmittance. Kinetics mode will collect absorbance data at a particular wavelength versus time. 
• For measurement of Absorbance / Transmittance: 
• Once the UV Probe software is up and running, click on the “Photometric” button on the toolbar. 
• Click on the “Method” button on the toolbar to set up the conditions of the photometric study. A new window will pop up. Set the wavelength, scan speed, and interval and input the desired information. 
• Load solvent into both sample compartments in the UV-Vis and press “baseline”. 
• Fill the cuvettes with blank solution and place it in both sample & reference holders & press Auto Zero the current wavelength will automatically be set to `0’ Abs. (100% T). 
• Then remove the front side cuvette, and discard the blank solution. Rinse the cuvette with standard/sample solution. Then fill the cuvette with standard/sample solution & replace it in the cell holder. The monitor will display the Absorbance of the standard/sample solution at the wavelength displayed. 
• Press “start” to take standard & sample absorbance. 
• After absorbance has been collected, click on “File, Save As” in order to permanently save the absorbance file to the PC.

For measurement of Spectrum

• Once the UV Probe software is up and running, click on the “Spectrum” button on the toolbar. 
• The spectrophotometer should be zeroed out (baseline correction) already. If not go back and re-do the baseline correction on the instrument. 
• Place the sample and diluent solvent cuvettes in respective cell holders, close the cover, and click on the “Spectrum” button on the toolbar. Click on the “Method” button on the toolbar to set up the conditions of the spectrum study. A new window will pop up. Input the desired wavelength range, scan speed, and scan mode. 
• Now Click ‘Start’ at the bottom of the UV Probe window, and the graph starts to form for the absorption spectrum. This graph shows the amount of light being absorbed by the sample for the wavelengths between the selected wavelengths. 
• After the spectrum has been collected, click on “File, Save As” in order to permanently save the spectrum file to the PC. 
• Label peaks on the spectrum: Click on the Peak Pick button on the toolbar. The Peak Pick table will then appear on the left side of the screen. Right-click the mouse on the Peak Pick table, and left-click on Properties. Now change parameters, such as the threshold for labeling peaks, whether the graph is labeled with the peak table number or the actual absorbance value, and whether peaks or valleys are labeled. 
• After setting up the Peak Pick table, click on the Peak Pick table to make it the active window. Then click on File, Print Preview. The report generator will show you how the printout will appear. If it looks ok, then click on Print.

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For measurement of Kinetic Study

• Once the UV Probe software is up and running, click on the “Kinetic” button on the toolbar. 
• Click on the “Method” button on the toolbar to set up the conditions of the kinetic study. A new window will pop up. Input the desired information. 
• The wavelength to be monitored. 
• “Timing Mode” should be set to manual. 
• Units = seconds 
• Cycle Time = how often to record the absorbance of the solution. 
• Number of Reading = self-explanatory 
• No need to change anything else. Click “OK” to measure the kinetic study. 
• Fill a cuvette with the solvent used and place the cuvette in the reference holder. 
• Place one part of the reaction solution in the sample cuvette. 
• Place the other part of the reaction solution in the sample cuvette. The reaction has started. 
• Quickly start the timer 
• Using parafilm seal up the sample cuvette. Shake the sample cuvette two or three times. Remove the parafilm. 
• Place the sample cuvette in the sample holder in the UV Spectrophotometer and close the lid. 
• Click on the “Start” button on the bottom toolbar. 
• Stop the Timer 
• Kinetic data will be collected over the period of time specified, when the data collection is done save the data.

Exit the PC Control Mode

• Be sure to save all data collected. Then click on the Disconnect button near the bottom of the screen. Exit from the UV Probe software. 
• Press the “Return” key to return local control to the spectrometer. This allows the instrument to turn off the deuterium lamp. 
• Turn `OFF’ the power switch located on the left-hand side of the UV Spectrophotometer. 

ABBREVIATIONS

UV – Ultra Violet 

PC – Program Controller 

T - Transmittance 

QC – Quality Control 

QA – Quality Assurance

ANNEXURE

Nil

REVISION HISTORY

Nil

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