Calculating % Transmission from Energy Spectra

 

Calculating Transmission Spectra from the E1 and E2 Energy Spectra

Using a PerkinElmer Lambda spectrometer, it is possible to calculate the transmission spectra (uncorrected) from the E1 (sample) and E2 (reference) energy spectra. The procedure to do this is described herein. A PerkinElmer Lambda 1050 running UVWinlab V6 is used for this procedure example.

The first step in the procedure requires the user to determine the correct PMT energy setting required to operate properly in the E1 and E2 modes.
After power up, and loading of UVWinlab, from UVWinlab Explorer, click on the Instrument menu, and then click on the Lambda instrument icon and then click on the Manual Control icon.
Using the Manual Control instrument settings enter a wavelength of 520 nm, a 2 nm slit, and then set the ordinate mode to E1 – then click on the Apply button. The maximum energy in the UV/Vis range is typically around 520 nm. The wavelength of maximum UV energy is typically around 250 nm, but this will be less than the signal at 520 nm.
Observe the E1 signal level on the live display. If the energy is between 95 and 100, the detector will be saturated. The energy needs to be dropped. The energy level is controlled by the PMT Gain setting. The default is 30. Enter a value of 15 to start and then click on Apply. The value should drop somewhere in the 10 to 30 range. Note that the bandpass selected will have an effect on the displayed E1 signal level, as well as the type of detector module being used. A PMT gain of 15 can be set for the standard detector module, and a slit of 2 nm.
After the PMT gain is determined, close the Manual Control mode, and click on a Scan method. Set an ordinate mode to E1, and the PMT Gain determined to be correct (i.e., 15). In the example shown here, a scan range from 700 to 400 nm was set, E1, and a PMT Gain of 15.



Click on the Sample Info menu in the method, and enter three sample names, called… E1_Raw E2_Raw E1_Sample
Click on Start to collect E1_Raw. When prompted for the next sample, click on Cancel, return to the Data Collection screen, and enter E2 for the ordinate mode. Click on Start to collect E2_Raw. An example of the overlaid E1 and E2 spectra is shown below. It is important not to change any other settings between the E1 and E2 scans.

The ratio of (E1/E2)*100 is the uncorrected transmission baseline. This spectrum can be calculated in UVWinlab V6 by clicking on the Processing section, and then adding an equation. For Equation 1, click to set the equation (E1_Raw/E2_Raw)*100.

Spectra directly calculated with equations will be placed into the Results section, under Custom. For each equation the spectra will be sequentially named Equation1, Equation2, etc. The calculated uncorrected transmission spectrum can be view there. An example is below.

To scan a sample in the energy mode, insert the sample into the sample side cell holder. Set the ordinate mode to E1. Leave all other Data Collection settings intact. Click on Start. The example below is a holmium oxide filter scanned in the E1 mode overlaid with the E1_Raw curve.

To calculate the E1 sample curve, in Processing, and another equation (Equation 2) to subtract E1_Sample from E1_Raw. See example. Again, the calculated spectrum will be placed in to Custom tab under Results, called Equation2.Sample. The curve calculated Equation 2 is shown below.

To calculate the transmission spectrum, the spectrum from Equation 2 needs to be ratioed to E2_Raw. In processing, add another equation (Equation 3) and set the final calculation as… [1-[Equation2/E2_Raw]]*100

This will calculate an uncorrected transmission spectrum and place the result into the Custom tab. Shown below is the E1 and E2 calculated transmission of holmium oxide, overlaid with the corrected spectra of holmium oxide scanned in the normal %T mode.