31 August 2022 0 8K Report

Hello everyone, I am new to liposome characterization and hope to get your insights about an experiment I am running. I took UV-vis spectra for POPC liposomes (undiluted and a set of dilutions 1:1, 1:10, 1:25) using Quartz cuvettes and a Cary 6000i UV-Vis-NIR Spectrophotometer and got the attached results. I think the peak ~205 nm is the one of interest based on the literature, and I see a dose-dependent signal in both trials (attached Fig A and B). But I also have quite a few concerns about the data.

  • I am not sure whether I took the 100% and 0% transmittance correctly while setting the baseline using only solvent PBS. There was no difference between the baselines, blue 100%, red 0% (in Fig C in the Powerpoint). I expected a decrease between 100% and 0%. I tried to see the beam turning off the room light, but I could not see it on the cardboard that I was using to block it. How to get the 0% correctly? I had the lamp on for 20 minutes before starting the experiment.
  • I tried my samples with both single front (same cuvette-Fig A) and double mode (two cuvettes-Fig B). And I saw a different amount of noise in the data. Which mode is the preferred mode?
  • Followup to the previous question: Is it normal to get the amount of noise in the data I have between the 190-200 nm range? For the single front mode, I also have noise between 340-440 nm (in the second slide), which I don't have in the double beam mode. What causes this, and how to avoid it?
  • I saw the absorbance maxima can be set to 10. As a result, the peak T ~205 nm in the second trial looks flat or tapered. Also, the precise position of the peak seems to shift between the two trials. I didn't try changing averaging time or data interval. What should I possibly change here?

    • I would greatly appreciate any help in troubleshooting this data! Thank you so much!

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