Lambda 35 UV-VIS Standard Operating Procedure

Primary Equipment:
Created: June 3, 2011 by Robert Cox

Revised: January 7, 2014 by Robert Cox

University of Utah, Chemical Engineering

P.E. Lambda 35 UV/VIS Spectrophotometer

Standard Operating Procedure 5/2011 R.Cox


Check in with the lab manager prior to operating this equipment.

Polystyrene cuvettes are used for most analysis. Do not use polystyrene cuvettes for acetone.

Disposable polystyrene cuvettes are not resistant to organic solvents, thus

non-water based samples should be analyzed in glass or quartz cuvettes.

If you require cuvettes of a different material, check with the lab managers or bring your own.

This SOP outlines the basic operation of the Lambda 35 using the Winlab software. For information that is more detailed consult the help files and the software manual.

The default data path is C:uvwinlabdatastudent.


Wavelength range 190 – 1100nm

Bandwidth 0.5, 1,2,4 nm, variable


Safety glasses must be worn at all times while working in the lab.

Wear gloves when handling chemicals.

Never open any part of the UV-Vis except the sample compartment. There are dangerous levels of UV radiation inside.

Start up

1. Log on to the PC with the user name and password posted on the monitor.

2. Open the sample compartment cover on the Lambda 35 UV-VIS, make sure the beam path is free, that is, no sample or accessories are in the path.

3. Close the cover.

4. Turn on the spectrophotometer using the green switch located on the top right rear corner.

5. Although not critical, for optimum performance, you should leave the instrument switched on for ~ 30 minutes to allow the lamps to stabilize. After warm up, re-initialize the calibration sequence by toggling the power switch off, wait a moment and then toggle the power switch on. Wait until all initialization is complete before starting the software (UV WinLab).

6. A note about methods:

The UV-VIS is controlled by “methods”. This document describes two method types, scan and concentration. In the methods window, scan methods are accessed via the SCAN tab, concentration methods are accessed via the CONC tab. The method defines a set of parameters that control the instrument. If you are using the UV-VIS for the first time and you want to scan across a range of wavelengths, it is recommended that you start with the 000MASTER.MSC method. This method performs a scan across the entire bandwidth of the instrument, 190 – 1100nm.

Once you complete a scan with this method, you can alter the method as required and then save your new method using a new name.

If you would like to scan for concentration refer to the section on running a concentration method.

Running a Scan Method:

1. Double click on the Lambda 35 icon on the desktop.

2. From the methods window, double click on the 00MASTER.MSC method.

3. On the bottom of the methods window, you will see three tabs: Scan, Inst and Sample. Select the SCAN tab.

Scan settings:

4. Start and end wavelength: The default setting is 1,110 – 190, this is the full range of the instrument. If you are uncertain where your peaks of interest will be it is recommended to run the full scan, you can narrow the scan later and save the method under a new name.

The following default settings are recommended:

5. Number of cycles = 1, Data interval = 1.

6. Autosave = On, Autoprint = Off, Autolist = On

7. Ordinate max = 7, min = 0

8. Set the display settings as desired, the default is Overlay.

9. If you save the method under a new name, enter new method info as desired.

Instrument Tab Settings:

10. Ordinate mode = A, lamps = on, slit = 2.0, scan speed = 960, smooth = 0

Lamp change = 326 nm

Sample Tab Settings:

11. Enter a results filename that ends in a number, it will be auto-incremented as you run more samples.

12. Enter the number of samples you expect to run, label the sample data and ID as desired.

13. Set the calculation factor to one, see the help file for further information.

14. When saving the method under a new name always check the “Autozero on start” and “Use next autoinc, filename”.

15. This completes the method setup.

Scanning a sample:

1. Verify that there are no samples in the instrument.

2. Place a polystyrene cuvette containing DI water or your reference solvent in the reference cell (rear). Close the cover.

3. Click “start”. The instrument auto zeros then a blank sample is requested.

4. Place another cuvette containing DI water or your blank of choice in the sample cell holder (front) and close the cover.

5. Click “OK”. Background correction is performed (which takes a while) and then the next “sample” is requested.

6. Remove the “blank” from the sample cell holder (front) and insert your sample cuvette. The reference cell remains in the rear position. Close the cover.

7. Click “OK”. When the scan is complete, the spectrum is displayed in the Graph 1 window.

8. The numerical concentration results are displayed in the results window. Copy and paste these results into your word or excel document.

9. The spectral results are displayed in the results window. Select the spectra tab then select the spectra of interest. Copy and paste the spectra to your document.

To view and save spectra:

Spectra are saved as .SP files

1. Open the data region window

2. Select the spectra tab

3. Open the file displayed in the spectra window.

4. The graph should be displayed.

5. Go to file “save” to save the graph and associated data file. You must save the graph before the graph and data files will list in the directory.

6. Copy and paste the graph as desired.

7. To switch the X / Y axis select data handling then select AT.

8. Review the icons located at the top of the window for more display options.

To retrieve numerical data:

1. Numerical data files are .rls type files, they consist of a list of abscissa and ordinate values.

2. With the graph window open and the graph of your data on display, select data handling from the top menu, then select arithmetic.

3. The arithmetic window opens and within it are the graph and results windows.

4. Close the graph window.

5. With the results window active, select file – open. All of the rls files in the selected folder should be visible. Open your results file, copy and paste the data as desired.

To label peaks:

1. Go to view – label peaks

2. Select label peak: Display both

3. To insure all peaks are labeled adjust the threshold value down as needed, a good starting point for the threshold is 0.01.

4. Click okay, you should see the plot with peaks labeled.

5. If the highest peak is not labeled, expand the y-axis, ordinate range.

6. Refer to the help files for further information.

7. To redraw the graph use the tools at the top, right side of the graph window.

8. The tool functions are displayed at the bottom of the window.

9. For example; if the data is bunched together go to the X Y tool and change the abscissa range.

10. Copy and paste your results to a word or excel document as desired.

Running a Concentration Method:

1. From the main Winlab window, select the CONC tab at the bottom of the methods window.

2. Open the CONCM1.MCO method.

Measurement page settings:

3. On the measurement page set the ordinate mode as required, typically you will set the ordinate mode to single wavelength in order to determine the concentration at that wavelength.

Concentration tab settings:

4. No Concentration

5. Cone = user

6. User = 1

7. Set wavelengths as desired

8. Set Replicates to 1, # of samples = 1

9. Enter method info and save under a new name

Instrument tab settings:

10. Response = 0.1

11. Scan speed = 960

12. Lamp UV = on, Lamp VIS = ON

13. Slit = 2.00, smooth = 0

14. Lamp change wavelength = 326.0 nm

15. Sample Tab

16. Enter results filename

17. Calc factor = factor

18. Enter # of samples

19. Enter sample ID, factor = 1, enter info as desired

Output tab settings:

20. Autosave = on, all others unchecked

21. Save file

22. Insert reference in rear cell

23. Select START

24. Follow the sample prompts to insert a blank in the rear � reference, sample compartment, insert the blank and click okay. The first “blank” scan is to auto zero the instrument. The prompts continue for the sample blank and samples for analysis.

25. When the scan is completed, the concentration results are displayed, copy and paste the results data as required.

To create a new method:

From the top tool bar select application:

Then select the type of method you would like to create: scan, conc, etc.

To create a scan method click on scan.

A dialog window will open and a message will be displayed saying “cannot open file” disregard this message.

Before defining the method parameters go to the top tool bar and select file – save,

Name and save the method.

Now proceed to define the method parameters.

Save the method once all parameters are defined.

1. In the file window, choose the type. Choose scan to start with.

2. Turn on auto save, change the start and end wavelength if necessary, and enter any method information in the dialog box at the bottom.

3. Click the “inst” tab. Change the ordinate mode if desired but the rest should not need to be changed.

4. Click the “sample” tab. Remember to put a number after the filename or your previous file will be over-written. Enter the number of samples you will be running in the dialog box. Change the “result filename” and the “sample identity” for each sample.

5. Click “save as” and enter a file name in the “filename” dialog box.

a. Select “Auto zero on start”.

6. Click “Use next autoinc.filename” to automatically save with filename plus three digits.

Shut Down

1. Exit the UV WinLab program. Do not turn off the computer.

2. Open the sample compartment cover and remove samples and cells.

3. Label and properly store any chemicals you plan on keeping in the lab.

4. Clean up cells and any spills.

5. Close the sample compartment cover.

6. Switch off the spectrophotometer.