I'm enquiring about o-phthalaldehyde modified colorimetric method in quantifying cholesterol for lowering-cholesterol ability of probiotics. I only know about experiment steps but not the rules. I will sumarize those ones:
1 ml supernatant + KOH+ abs alcohol -> vortex-> heat in 37oC -> add distilled water and hexane -> transfer hexane layer into a glass tube -> evaporate under nitrogen gas -> resolve with o-phthalaldehyde reagent (The reagent contained 0.5 mg of o-phthalaldehyde per milliliter of glacial acetic acid) -> stand in 10 min -> add sulfuric acid -> vortex -> read absorbance.
There are some point I really don't understand: why we evaporate under nitrogen gas, and what sulfuric acid is for, and the glacial acetic acid.
Could someone help me answer these or show me the source to work out them?
Dear,
The hexane can be evaporated under nitrogen otherwise you can evaporate it on room temperature which would take longer time. You can use speed vacuum concentrator to evaporate it otherwise as well. The glacial acetic acid is good solvent for ortho-phthalaldehyde. The sulfuric acid reacts with the reaction product and gives final pink coloured product which is read on a spectrophotometer.
Regards,
A K Dubey
Hi, you can see this link I hope its useful
http://scialert.net/fulltext/?doi=ijbc.2012.37.41&org=10
https://www.ncbi.nlm.nih.gov/pubmed/14580182
Following on this topic, I am trying this method but when preparing the o-phthalaldehyde solution in acetic acid, it does not change color. Any leads as to why this can be happening? Using a 0.5mg/mL as final concentration.
Thanks
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