December 11, 2019, 12:05:13 PM
Forum Rules: Read This Before Posting


Topic: Enzyme Activity Assay - Can I use the Colorimeter I Have? (Optics)  (Read 122 times)

0 Members and 1 Guest are viewing this topic.

Offline anonymous10012

  • Regular Member
  • ***
  • Posts: 11
  • Mole Snacks: +1/-0
Hello,

Thank you for clicking on my post. I am trying to better understand the relationship between absorbance and reflectance (not transmittance). I have included a link to a conversation on researchgate.

https://www.researchgate.net/post/Can_I_convert_reflectance_to_absorbance

I want to measure peroxidase activity of a pretty translucent aqueous extract of horseradish. The colorimeter that I have only measures reflectance (it was purchased for a different purpose).

Can anyone weigh in on whether or not the conversion A=log 1/R would suffice in this application?

Here is another discussion about it. Optics is a weak point for me. Thank you!

https://www.researchgate.net/post/Is_there_a_conversion_from_Reflectance_to_Transmittance_in_spectroscopy

Offline Corribus

  • Chemist
  • Sr. Member
  • *
  • Posts: 2784
  • Mole Snacks: +443/-20
  • Gender: Male
  • A lover of spectroscopy and chocolate.
Re: Enzyme Activity Assay - Can I use the Colorimeter I Have? (Optics)
« Reply #1 on: November 25, 2019, 10:37:44 AM »
Theoretically you can relate reflectance measurements to absorption measurements. This is frequently done on solid samples using a device called an integrating sphere.

https://www.newport.com/t/integrating-sphere-fundamentals-and-applications

Your problem is that if the reflectance isn't truly diffuse, converting your reflectance to absorbance won't give you a representation of the bulk absorbance properties of your sample. Moreover the reflectance measurement usually needs to be calibrated against a material with a known standard reflectance, presumably to account for sensitivity variations in your instrument.

Could you do this with a handheld reflectance colorimeter? Maybe.... but it's hard to get good absorbance measurements from an integrating sphere, which is intended for this purposes. Because of that, I don't know that I'd trust any absorbance measurement made from a reflectance colorimeter, certainly not a quantitative measurement.

UV Spectrophotometers are cheap and pretty ubiquitous - can't you find one somewhere to use for your measurement?

ALSO: Please don't cross-post.
What men are poets who can speak of Jupiter if he were like a man, but if he is an immense spinning sphere of methane and ammonia must be silent?  - Richard P. Feynman

Sponsored Links