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Chemistry Forums for Students => Physical Chemistry Forum => Topic started by: anonymous10012 on November 25, 2019, 10:18:31 AM

Title: Enzyme Activity Assay - Can I use the Colorimeter I Have? (Optics)
Post by: anonymous10012 on November 25, 2019, 10:18:31 AM
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
Title: Re: Enzyme Activity Assay - Can I use the Colorimeter I Have? (Optics)
Post by: Corribus 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.